US20020160941A1 - Treating compositions with lactoferrin - Google Patents
Treating compositions with lactoferrin Download PDFInfo
- Publication number
- US20020160941A1 US20020160941A1 US10/023,096 US2309601A US2002160941A1 US 20020160941 A1 US20020160941 A1 US 20020160941A1 US 2309601 A US2309601 A US 2309601A US 2002160941 A1 US2002160941 A1 US 2002160941A1
- Authority
- US
- United States
- Prior art keywords
- lactoferrin
- ala
- leu
- gly
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010063045 Lactoferrin Proteins 0.000 title claims abstract description 164
- 102000010445 Lactoferrin Human genes 0.000 title claims abstract description 152
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 title claims abstract description 143
- 229940078795 lactoferrin Drugs 0.000 title claims abstract description 143
- 235000021242 lactoferrin Nutrition 0.000 title claims abstract description 143
- 239000000203 mixture Substances 0.000 title claims abstract description 14
- 230000000813 microbial effect Effects 0.000 claims abstract description 27
- 238000011109 contamination Methods 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims description 53
- 229910052751 metal Inorganic materials 0.000 claims description 13
- 239000002184 metal Substances 0.000 claims description 13
- 235000013305 food Nutrition 0.000 claims description 10
- 238000011068 loading method Methods 0.000 claims description 10
- 230000000979 retarding effect Effects 0.000 claims description 3
- 235000013622 meat product Nutrition 0.000 claims 10
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 82
- 229910052742 iron Inorganic materials 0.000 description 41
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 39
- 108090000623 proteins and genes Proteins 0.000 description 36
- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 description 31
- 102000050459 human LTF Human genes 0.000 description 28
- 108020004414 DNA Proteins 0.000 description 27
- 239000000463 material Substances 0.000 description 26
- 239000002299 complementary DNA Substances 0.000 description 24
- 239000013612 plasmid Substances 0.000 description 24
- 239000000499 gel Substances 0.000 description 23
- 238000011282 treatment Methods 0.000 description 23
- 239000012634 fragment Substances 0.000 description 22
- 102000004169 proteins and genes Human genes 0.000 description 22
- 235000018102 proteins Nutrition 0.000 description 21
- 239000000872 buffer Substances 0.000 description 20
- 239000011780 sodium chloride Substances 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 16
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 15
- 238000000746 purification Methods 0.000 description 14
- 238000004587 chromatography analysis Methods 0.000 description 13
- 239000003463 adsorbent Substances 0.000 description 12
- 238000001042 affinity chromatography Methods 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 12
- 108091008146 restriction endonucleases Proteins 0.000 description 12
- 230000002421 anti-septic effect Effects 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 10
- 239000007995 HEPES buffer Substances 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 108091034117 Oligonucleotide Proteins 0.000 description 9
- 108010076504 Protein Sorting Signals Proteins 0.000 description 9
- 239000011543 agarose gel Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 150000007523 nucleic acids Chemical class 0.000 description 9
- 239000004593 Epoxy Substances 0.000 description 8
- 230000029087 digestion Effects 0.000 description 8
- 238000001990 intravenous administration Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 7
- 229920000936 Agarose Polymers 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 239000005289 controlled pore glass Substances 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 239000007974 sodium acetate buffer Substances 0.000 description 7
- 238000011200 topical administration Methods 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- 102000008133 Iron-Binding Proteins Human genes 0.000 description 6
- 108010035210 Iron-Binding Proteins Proteins 0.000 description 6
- 241000235058 Komagataella pastoris Species 0.000 description 6
- 239000007990 PIPES buffer Substances 0.000 description 6
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 6
- 108010041407 alanylaspartic acid Proteins 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 6
- 235000011130 ammonium sulphate Nutrition 0.000 description 6
- 108010092854 aspartyllysine Proteins 0.000 description 6
- 108010049041 glutamylalanine Proteins 0.000 description 6
- 235000020256 human milk Nutrition 0.000 description 6
- 210000004251 human milk Anatomy 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 108010015796 prolylisoleucine Proteins 0.000 description 6
- 235000015872 dietary supplement Nutrition 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000012847 fine chemical Substances 0.000 description 5
- 210000004293 human mammary gland Anatomy 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 230000000717 retained effect Effects 0.000 description 5
- 229920006395 saturated elastomer Polymers 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- SBKVPJHMSUXZTA-MEJXFZFPSA-N (2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-5-amino-2-[[2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(1H-indol-3-yl)propanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-5-oxopentanoyl]pyrrolidine-2-carbonyl]amino]-4-methylsulfanylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 SBKVPJHMSUXZTA-MEJXFZFPSA-N 0.000 description 4
- 208000030507 AIDS Diseases 0.000 description 4
- 108010025188 Alcohol oxidase Proteins 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- HDNXXTBKOJKWNN-WDSKDSINSA-N Gly-Glu-Asn Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O HDNXXTBKOJKWNN-WDSKDSINSA-N 0.000 description 4
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 4
- 101150069554 HIS4 gene Proteins 0.000 description 4
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 4
- IBMVEYRWAWIOTN-RWMBFGLXSA-N Leu-Arg-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(O)=O IBMVEYRWAWIOTN-RWMBFGLXSA-N 0.000 description 4
- 108010038049 Mating Factor Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 4
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 4
- JFAWZADYPRMRCO-UBHSHLNASA-N Val-Ala-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JFAWZADYPRMRCO-UBHSHLNASA-N 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000356 anti-lactoferrin effect Effects 0.000 description 4
- 108010068380 arginylarginine Proteins 0.000 description 4
- 108010062796 arginyllysine Proteins 0.000 description 4
- 108010038633 aspartylglutamate Proteins 0.000 description 4
- 108010047857 aspartylglycine Proteins 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 108010034529 leucyl-lysine Proteins 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 229910021645 metal ion Inorganic materials 0.000 description 4
- 239000002674 ointment Substances 0.000 description 4
- 108010053725 prolylvaline Proteins 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 108010061238 threonyl-glycine Proteins 0.000 description 4
- 230000000699 topical effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 241000235648 Pichia Species 0.000 description 3
- 206010035664 Pneumonia Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- YKCWQPZFAFZLBI-UHFFFAOYSA-N cibacron blue Chemical compound C1=2C(=O)C3=CC=CC=C3C(=O)C=2C(N)=C(S(O)(=O)=O)C=C1NC(C=C1S(O)(=O)=O)=CC=C1NC(N=1)=NC(Cl)=NC=1NC1=CC=CC=C1S(O)(=O)=O YKCWQPZFAFZLBI-UHFFFAOYSA-N 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002324 mouth wash Substances 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- -1 nickel or copper Chemical compound 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 2
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 2
- SDMAQFGBPOJFOM-GUBZILKMSA-N Ala-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SDMAQFGBPOJFOM-GUBZILKMSA-N 0.000 description 2
- IMMKUCQIKKXKNP-DCAQKATOSA-N Ala-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCN=C(N)N IMMKUCQIKKXKNP-DCAQKATOSA-N 0.000 description 2
- TTXMOJWKNRJWQJ-FXQIFTODSA-N Ala-Arg-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N TTXMOJWKNRJWQJ-FXQIFTODSA-N 0.000 description 2
- LSLIRHLIUDVNBN-CIUDSAMLSA-N Ala-Asp-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LSLIRHLIUDVNBN-CIUDSAMLSA-N 0.000 description 2
- NWVVKQZOVSTDBQ-CIUDSAMLSA-N Ala-Glu-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NWVVKQZOVSTDBQ-CIUDSAMLSA-N 0.000 description 2
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 2
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 2
- SIGTYDNEPYEXGK-ZANVPECISA-N Ala-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@@H](N)C)C(O)=O)=CNC2=C1 SIGTYDNEPYEXGK-ZANVPECISA-N 0.000 description 2
- GSHKMNKPMLXSQW-KBIXCLLPSA-N Ala-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C)N GSHKMNKPMLXSQW-KBIXCLLPSA-N 0.000 description 2
- AUFACLFHBAGZEN-ZLUOBGJFSA-N Ala-Ser-Cys Chemical compound N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O AUFACLFHBAGZEN-ZLUOBGJFSA-N 0.000 description 2
- ZVWXMTTZJKBJCI-BHDSKKPTSA-N Ala-Trp-Ala Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 ZVWXMTTZJKBJCI-BHDSKKPTSA-N 0.000 description 2
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 2
- XPSGESXVBSQZPL-SRVKXCTJSA-N Arg-Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XPSGESXVBSQZPL-SRVKXCTJSA-N 0.000 description 2
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 2
- BAVDUESNGSMLPI-CIUDSAMLSA-N Arg-Asn-Gly-Ser Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BAVDUESNGSMLPI-CIUDSAMLSA-N 0.000 description 2
- KMSHNDWHPWXPEC-BQBZGAKWSA-N Arg-Asp-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KMSHNDWHPWXPEC-BQBZGAKWSA-N 0.000 description 2
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 description 2
- GIMTZGADWZTZGV-DCAQKATOSA-N Arg-Lys-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N GIMTZGADWZTZGV-DCAQKATOSA-N 0.000 description 2
- GRRXPUAICOGISM-RWMBFGLXSA-N Arg-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O GRRXPUAICOGISM-RWMBFGLXSA-N 0.000 description 2
- AFNHFVVOJZBIJD-GUBZILKMSA-N Arg-Met-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O AFNHFVVOJZBIJD-GUBZILKMSA-N 0.000 description 2
- VUGWHBXPMAHEGZ-SRVKXCTJSA-N Arg-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N VUGWHBXPMAHEGZ-SRVKXCTJSA-N 0.000 description 2
- AMIQZQAAYGYKOP-FXQIFTODSA-N Arg-Ser-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O AMIQZQAAYGYKOP-FXQIFTODSA-N 0.000 description 2
- ADPACBMPYWJJCE-FXQIFTODSA-N Arg-Ser-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O ADPACBMPYWJJCE-FXQIFTODSA-N 0.000 description 2
- OQPAZKMGCWPERI-GUBZILKMSA-N Arg-Ser-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O OQPAZKMGCWPERI-GUBZILKMSA-N 0.000 description 2
- VDCIPFYVCICPEC-FXQIFTODSA-N Asn-Arg-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O VDCIPFYVCICPEC-FXQIFTODSA-N 0.000 description 2
- LJUOLNXOWSWGKF-ACZMJKKPSA-N Asn-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N LJUOLNXOWSWGKF-ACZMJKKPSA-N 0.000 description 2
- SQZIAWGBBUSSPJ-ZKWXMUAHSA-N Asn-Cys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)N)N SQZIAWGBBUSSPJ-ZKWXMUAHSA-N 0.000 description 2
- OKZOABJQOMAYEC-NUMRIWBASA-N Asn-Gln-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OKZOABJQOMAYEC-NUMRIWBASA-N 0.000 description 2
- IHUJUZBUOFTIOB-QEJZJMRPSA-N Asn-Gln-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N IHUJUZBUOFTIOB-QEJZJMRPSA-N 0.000 description 2
- PPMTUXJSQDNUDE-CIUDSAMLSA-N Asn-Glu-Arg Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PPMTUXJSQDNUDE-CIUDSAMLSA-N 0.000 description 2
- RAKKBBHMTJSXOY-XVYDVKMFSA-N Asn-His-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O RAKKBBHMTJSXOY-XVYDVKMFSA-N 0.000 description 2
- NLRJGXZWTKXRHP-DCAQKATOSA-N Asn-Leu-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NLRJGXZWTKXRHP-DCAQKATOSA-N 0.000 description 2
- YVXRYLVELQYAEQ-SRVKXCTJSA-N Asn-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N YVXRYLVELQYAEQ-SRVKXCTJSA-N 0.000 description 2
- JXMREEPBRANWBY-VEVYYDQMSA-N Asn-Thr-Arg Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JXMREEPBRANWBY-VEVYYDQMSA-N 0.000 description 2
- QNNBHTFDFFFHGC-KKUMJFAQSA-N Asn-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O QNNBHTFDFFFHGC-KKUMJFAQSA-N 0.000 description 2
- GHWWTICYPDKPTE-NGZCFLSTSA-N Asn-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N GHWWTICYPDKPTE-NGZCFLSTSA-N 0.000 description 2
- XBQSLMACWDXWLJ-GHCJXIJMSA-N Asp-Ala-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XBQSLMACWDXWLJ-GHCJXIJMSA-N 0.000 description 2
- CXBOKJPLEYUPGB-FXQIFTODSA-N Asp-Ala-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(=O)O)N CXBOKJPLEYUPGB-FXQIFTODSA-N 0.000 description 2
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 2
- XACXDSRQIXRMNS-OLHMAJIHSA-N Asp-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N)O XACXDSRQIXRMNS-OLHMAJIHSA-N 0.000 description 2
- IAMNNSSEBXDJMN-CIUDSAMLSA-N Asp-Cys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)O)N IAMNNSSEBXDJMN-CIUDSAMLSA-N 0.000 description 2
- KVPHTGVUMJGMCX-BIIVOSGPSA-N Asp-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)[C@H](CC(=O)O)N)C(=O)O KVPHTGVUMJGMCX-BIIVOSGPSA-N 0.000 description 2
- HSWYMWGDMPLTTH-FXQIFTODSA-N Asp-Glu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HSWYMWGDMPLTTH-FXQIFTODSA-N 0.000 description 2
- RRKCPMGSRIDLNC-AVGNSLFASA-N Asp-Glu-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RRKCPMGSRIDLNC-AVGNSLFASA-N 0.000 description 2
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 2
- YWLDTBBUHZJQHW-KKUMJFAQSA-N Asp-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)O)N YWLDTBBUHZJQHW-KKUMJFAQSA-N 0.000 description 2
- GYWQGGUCMDCUJE-DLOVCJGASA-N Asp-Phe-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(O)=O GYWQGGUCMDCUJE-DLOVCJGASA-N 0.000 description 2
- WMLFFCRUSPNENW-ZLUOBGJFSA-N Asp-Ser-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O WMLFFCRUSPNENW-ZLUOBGJFSA-N 0.000 description 2
- HRVQDZOWMLFAOD-BIIVOSGPSA-N Asp-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N)C(=O)O HRVQDZOWMLFAOD-BIIVOSGPSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 2
- MBPKYKSYUAPLMY-DCAQKATOSA-N Cys-Arg-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O MBPKYKSYUAPLMY-DCAQKATOSA-N 0.000 description 2
- UYYZZJXUVIZTMH-AVGNSLFASA-N Cys-Glu-Phe Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O UYYZZJXUVIZTMH-AVGNSLFASA-N 0.000 description 2
- KCSDYJSCUWLILX-BJDJZHNGSA-N Cys-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CS)N KCSDYJSCUWLILX-BJDJZHNGSA-N 0.000 description 2
- KCPOQGRVVXYLAC-KKUMJFAQSA-N Cys-Leu-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CS)N KCPOQGRVVXYLAC-KKUMJFAQSA-N 0.000 description 2
- KJJASVYBTKRYSN-FXQIFTODSA-N Cys-Pro-Asp Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CS)N)C(=O)N[C@@H](CC(=O)O)C(=O)O KJJASVYBTKRYSN-FXQIFTODSA-N 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 2
- NNQHEEQNPQYPGL-FXQIFTODSA-N Gln-Ala-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O NNQHEEQNPQYPGL-FXQIFTODSA-N 0.000 description 2
- DLOHWQXXGMEZDW-CIUDSAMLSA-N Gln-Arg-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O DLOHWQXXGMEZDW-CIUDSAMLSA-N 0.000 description 2
- LPYPANUXJGFMGV-FXQIFTODSA-N Gln-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N LPYPANUXJGFMGV-FXQIFTODSA-N 0.000 description 2
- PNENQZWRFMUZOM-DCAQKATOSA-N Gln-Glu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O PNENQZWRFMUZOM-DCAQKATOSA-N 0.000 description 2
- VOLVNCMGXWDDQY-LPEHRKFASA-N Gln-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N)C(=O)O VOLVNCMGXWDDQY-LPEHRKFASA-N 0.000 description 2
- FGYPOQPQTUNESW-IUCAKERBSA-N Gln-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N FGYPOQPQTUNESW-IUCAKERBSA-N 0.000 description 2
- TWIAMTNJOMRDAK-GUBZILKMSA-N Gln-Lys-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O TWIAMTNJOMRDAK-GUBZILKMSA-N 0.000 description 2
- XUMFMAVDHQDATI-DCAQKATOSA-N Gln-Pro-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XUMFMAVDHQDATI-DCAQKATOSA-N 0.000 description 2
- RWQCWSGOOOEGPB-FXQIFTODSA-N Gln-Ser-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O RWQCWSGOOOEGPB-FXQIFTODSA-N 0.000 description 2
- YADSXULAFMJZRL-QEJZJMRPSA-N Gln-Trp-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)N)N YADSXULAFMJZRL-QEJZJMRPSA-N 0.000 description 2
- SJMJMEWQMBJYPR-DZKIICNBSA-N Gln-Tyr-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CCC(=O)N)N SJMJMEWQMBJYPR-DZKIICNBSA-N 0.000 description 2
- NADWTMLCUDMDQI-ACZMJKKPSA-N Glu-Asp-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N NADWTMLCUDMDQI-ACZMJKKPSA-N 0.000 description 2
- RQNYYRHRKSVKAB-GUBZILKMSA-N Glu-Cys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O RQNYYRHRKSVKAB-GUBZILKMSA-N 0.000 description 2
- RAUDKMVXNOWDLS-WDSKDSINSA-N Glu-Gly-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O RAUDKMVXNOWDLS-WDSKDSINSA-N 0.000 description 2
- XMPAXPSENRSOSV-RYUDHWBXSA-N Glu-Gly-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XMPAXPSENRSOSV-RYUDHWBXSA-N 0.000 description 2
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-N 0.000 description 2
- MFNUFCFRAZPJFW-JYJNAYRXSA-N Glu-Lys-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MFNUFCFRAZPJFW-JYJNAYRXSA-N 0.000 description 2
- AQNYKMCFCCZEEL-JYJNAYRXSA-N Glu-Lys-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AQNYKMCFCCZEEL-JYJNAYRXSA-N 0.000 description 2
- PMSDOVISAARGAV-FHWLQOOXSA-N Glu-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 PMSDOVISAARGAV-FHWLQOOXSA-N 0.000 description 2
- KIEICAOUSNYOLM-NRPADANISA-N Glu-Val-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KIEICAOUSNYOLM-NRPADANISA-N 0.000 description 2
- JUGQPPOVWXSPKJ-RYUDHWBXSA-N Gly-Gln-Phe Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JUGQPPOVWXSPKJ-RYUDHWBXSA-N 0.000 description 2
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 2
- SCWYHUQOOFRVHP-MBLNEYKQSA-N Gly-Ile-Thr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SCWYHUQOOFRVHP-MBLNEYKQSA-N 0.000 description 2
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 description 2
- LOEANKRDMMVOGZ-YUMQZZPRSA-N Gly-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O LOEANKRDMMVOGZ-YUMQZZPRSA-N 0.000 description 2
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 2
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 2
- IBYOLNARKHMLBG-WHOFXGATSA-N Gly-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IBYOLNARKHMLBG-WHOFXGATSA-N 0.000 description 2
- LBDXVCBAJJNJNN-WHFBIAKZSA-N Gly-Ser-Cys Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(O)=O LBDXVCBAJJNJNN-WHFBIAKZSA-N 0.000 description 2
- 208000031856 Haemosiderosis Diseases 0.000 description 2
- NQKRILCJYCASDV-QWRGUYRKSA-N His-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 NQKRILCJYCASDV-QWRGUYRKSA-N 0.000 description 2
- VYUXYMRNGALHEA-DLOVCJGASA-N His-Leu-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O VYUXYMRNGALHEA-DLOVCJGASA-N 0.000 description 2
- MDOBWSFNSNPENN-PMVVWTBXSA-N His-Thr-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O MDOBWSFNSNPENN-PMVVWTBXSA-N 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- JRHFQUPIZOYKQP-KBIXCLLPSA-N Ile-Ala-Glu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O JRHFQUPIZOYKQP-KBIXCLLPSA-N 0.000 description 2
- FVEWRQXNISSYFO-ZPFDUUQYSA-N Ile-Arg-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FVEWRQXNISSYFO-ZPFDUUQYSA-N 0.000 description 2
- DCQMJRSOGCYKTR-GHCJXIJMSA-N Ile-Asp-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O DCQMJRSOGCYKTR-GHCJXIJMSA-N 0.000 description 2
- ZDNORQNHCJUVOV-KBIXCLLPSA-N Ile-Gln-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O ZDNORQNHCJUVOV-KBIXCLLPSA-N 0.000 description 2
- OONBGFHNQVSUBF-KBIXCLLPSA-N Ile-Gln-Cys Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(O)=O OONBGFHNQVSUBF-KBIXCLLPSA-N 0.000 description 2
- WZDCVAWMBUNDDY-KBIXCLLPSA-N Ile-Glu-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C)C(=O)O)N WZDCVAWMBUNDDY-KBIXCLLPSA-N 0.000 description 2
- ODPKZZLRDNXTJZ-WHOFXGATSA-N Ile-Gly-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N ODPKZZLRDNXTJZ-WHOFXGATSA-N 0.000 description 2
- DFFTXLCCDFYRKD-MBLNEYKQSA-N Ile-Gly-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N DFFTXLCCDFYRKD-MBLNEYKQSA-N 0.000 description 2
- 206010065973 Iron Overload Diseases 0.000 description 2
- 108010079091 KRDS peptide Proteins 0.000 description 2
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 2
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 2
- YOZCKMXHBYKOMQ-IHRRRGAJSA-N Leu-Arg-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N YOZCKMXHBYKOMQ-IHRRRGAJSA-N 0.000 description 2
- KKXDHFKZWKLYGB-GUBZILKMSA-N Leu-Asn-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKXDHFKZWKLYGB-GUBZILKMSA-N 0.000 description 2
- IIKJNQWOQIWWMR-CIUDSAMLSA-N Leu-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)N IIKJNQWOQIWWMR-CIUDSAMLSA-N 0.000 description 2
- PPTAQBNUFKTJKA-BJDJZHNGSA-N Leu-Cys-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PPTAQBNUFKTJKA-BJDJZHNGSA-N 0.000 description 2
- PPBKJAQJAUHZKX-SRVKXCTJSA-N Leu-Cys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CC(C)C PPBKJAQJAUHZKX-SRVKXCTJSA-N 0.000 description 2
- DLCXCECTCPKKCD-GUBZILKMSA-N Leu-Gln-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O DLCXCECTCPKKCD-GUBZILKMSA-N 0.000 description 2
- YFBBUHJJUXXZOF-UWVGGRQHSA-N Leu-Gly-Pro Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O YFBBUHJJUXXZOF-UWVGGRQHSA-N 0.000 description 2
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 2
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 2
- KYIIALJHAOIAHF-KKUMJFAQSA-N Leu-Leu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 KYIIALJHAOIAHF-KKUMJFAQSA-N 0.000 description 2
- ZGUMORRUBUCXEH-AVGNSLFASA-N Leu-Lys-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZGUMORRUBUCXEH-AVGNSLFASA-N 0.000 description 2
- BIZNDKMFQHDOIE-KKUMJFAQSA-N Leu-Phe-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=CC=C1 BIZNDKMFQHDOIE-KKUMJFAQSA-N 0.000 description 2
- PJWOOBTYQNNRBF-BZSNNMDCSA-N Leu-Phe-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)O)N PJWOOBTYQNNRBF-BZSNNMDCSA-N 0.000 description 2
- IZPVWNSAVUQBGP-CIUDSAMLSA-N Leu-Ser-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IZPVWNSAVUQBGP-CIUDSAMLSA-N 0.000 description 2
- JGKHAFUAPZCCDU-BZSNNMDCSA-N Leu-Tyr-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=C(O)C=C1 JGKHAFUAPZCCDU-BZSNNMDCSA-N 0.000 description 2
- FDBTVENULFNTAL-XQQFMLRXSA-N Leu-Val-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N FDBTVENULFNTAL-XQQFMLRXSA-N 0.000 description 2
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 2
- ZAWOJFFMBANLGE-CIUDSAMLSA-N Lys-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCCN)N ZAWOJFFMBANLGE-CIUDSAMLSA-N 0.000 description 2
- RLZDUFRBMQNYIJ-YUMQZZPRSA-N Lys-Cys-Gly Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)O)N RLZDUFRBMQNYIJ-YUMQZZPRSA-N 0.000 description 2
- BYEBKXRNDLTGFW-CIUDSAMLSA-N Lys-Cys-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O BYEBKXRNDLTGFW-CIUDSAMLSA-N 0.000 description 2
- OPTCSTACHGNULU-DCAQKATOSA-N Lys-Cys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCCN OPTCSTACHGNULU-DCAQKATOSA-N 0.000 description 2
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 2
- MYZMQWHPDAYKIE-SRVKXCTJSA-N Lys-Leu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O MYZMQWHPDAYKIE-SRVKXCTJSA-N 0.000 description 2
- TWPCWKVOZDUYAA-KKUMJFAQSA-N Lys-Phe-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O TWPCWKVOZDUYAA-KKUMJFAQSA-N 0.000 description 2
- MSSJJDVQTFTLIF-KBPBESRZSA-N Lys-Phe-Gly Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O MSSJJDVQTFTLIF-KBPBESRZSA-N 0.000 description 2
- AZOFEHCPMBRNFD-BZSNNMDCSA-N Lys-Phe-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=CC=C1 AZOFEHCPMBRNFD-BZSNNMDCSA-N 0.000 description 2
- CTJUSALVKAWFFU-CIUDSAMLSA-N Lys-Ser-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N CTJUSALVKAWFFU-CIUDSAMLSA-N 0.000 description 2
- WZVSHTFTCYOFPL-GARJFASQSA-N Lys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)N)C(=O)O WZVSHTFTCYOFPL-GARJFASQSA-N 0.000 description 2
- ULNXMMYXQKGNPG-LPEHRKFASA-N Met-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N ULNXMMYXQKGNPG-LPEHRKFASA-N 0.000 description 2
- OLWAOWXIADGIJG-AVGNSLFASA-N Met-Arg-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(O)=O OLWAOWXIADGIJG-AVGNSLFASA-N 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 2
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 2
- 208000006816 Neonatal Sepsis Diseases 0.000 description 2
- 101100068676 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) gln-1 gene Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 208000001388 Opportunistic Infections Diseases 0.000 description 2
- YMORXCKTSSGYIG-IHRRRGAJSA-N Phe-Arg-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N YMORXCKTSSGYIG-IHRRRGAJSA-N 0.000 description 2
- KAGCQPSEVAETCA-JYJNAYRXSA-N Phe-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N KAGCQPSEVAETCA-JYJNAYRXSA-N 0.000 description 2
- RLUMIJXNHJVUCO-JBACZVJFSA-N Phe-Gln-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 RLUMIJXNHJVUCO-JBACZVJFSA-N 0.000 description 2
- MPFGIYLYWUCSJG-AVGNSLFASA-N Phe-Glu-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 MPFGIYLYWUCSJG-AVGNSLFASA-N 0.000 description 2
- BIYWZVCPZIFGPY-QWRGUYRKSA-N Phe-Gly-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BIYWZVCPZIFGPY-QWRGUYRKSA-N 0.000 description 2
- KBVJZCVLQWCJQN-KKUMJFAQSA-N Phe-Leu-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KBVJZCVLQWCJQN-KKUMJFAQSA-N 0.000 description 2
- CJAHQEZWDZNSJO-KKUMJFAQSA-N Phe-Lys-Cys Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CS)C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CJAHQEZWDZNSJO-KKUMJFAQSA-N 0.000 description 2
- GPLWGAYGROGDEN-BZSNNMDCSA-N Phe-Phe-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O GPLWGAYGROGDEN-BZSNNMDCSA-N 0.000 description 2
- MCIXMYKSPQUMJG-SRVKXCTJSA-N Phe-Ser-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MCIXMYKSPQUMJG-SRVKXCTJSA-N 0.000 description 2
- XNMYNGDKJNOKHH-BZSNNMDCSA-N Phe-Ser-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XNMYNGDKJNOKHH-BZSNNMDCSA-N 0.000 description 2
- FQYUMYWMJTYZTK-UHFFFAOYSA-N Phenyl glycidyl ether Chemical compound C1OC1COC1=CC=CC=C1 FQYUMYWMJTYZTK-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- AMBLXEMWFARNNQ-DCAQKATOSA-N Pro-Asn-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@@H]1CCCN1 AMBLXEMWFARNNQ-DCAQKATOSA-N 0.000 description 2
- FUVBEZJCRMHWEM-FXQIFTODSA-N Pro-Asn-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O FUVBEZJCRMHWEM-FXQIFTODSA-N 0.000 description 2
- YFNOUBWUIIJQHF-LPEHRKFASA-N Pro-Asp-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)O)C(=O)N2CCC[C@@H]2C(=O)O YFNOUBWUIIJQHF-LPEHRKFASA-N 0.000 description 2
- LHALYDBUDCWMDY-CIUDSAMLSA-N Pro-Glu-Ala Chemical compound C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1)C(O)=O LHALYDBUDCWMDY-CIUDSAMLSA-N 0.000 description 2
- LGSANCBHSMDFDY-GARJFASQSA-N Pro-Glu-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N2CCC[C@@H]2C(=O)O LGSANCBHSMDFDY-GARJFASQSA-N 0.000 description 2
- WIPAMEKBSHNFQE-IUCAKERBSA-N Pro-Met-Gly Chemical compound CSCC[C@@H](C(=O)NCC(=O)O)NC(=O)[C@@H]1CCCN1 WIPAMEKBSHNFQE-IUCAKERBSA-N 0.000 description 2
- JLMZKEQFMVORMA-SRVKXCTJSA-N Pro-Pro-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 JLMZKEQFMVORMA-SRVKXCTJSA-N 0.000 description 2
- BGWKULMLUIUPKY-BQBZGAKWSA-N Pro-Ser-Gly Chemical compound OC(=O)CNC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 BGWKULMLUIUPKY-BQBZGAKWSA-N 0.000 description 2
- UIUWGMRJTWHIJZ-ULQDDVLXSA-N Pro-Tyr-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CCCCN)C(=O)O UIUWGMRJTWHIJZ-ULQDDVLXSA-N 0.000 description 2
- OOZJHTXCLJUODH-QXEWZRGKSA-N Pro-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 OOZJHTXCLJUODH-QXEWZRGKSA-N 0.000 description 2
- FIODMZKLZFLYQP-GUBZILKMSA-N Pro-Val-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FIODMZKLZFLYQP-GUBZILKMSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- HBOABDXGTMMDSE-GUBZILKMSA-N Ser-Arg-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O HBOABDXGTMMDSE-GUBZILKMSA-N 0.000 description 2
- GHPQVUYZQQGEDA-BIIVOSGPSA-N Ser-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)N)C(=O)O GHPQVUYZQQGEDA-BIIVOSGPSA-N 0.000 description 2
- COLJZWUVZIXSSS-CIUDSAMLSA-N Ser-Cys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N COLJZWUVZIXSSS-CIUDSAMLSA-N 0.000 description 2
- IXUGADGDCQDLSA-FXQIFTODSA-N Ser-Gln-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N IXUGADGDCQDLSA-FXQIFTODSA-N 0.000 description 2
- FMDHKPRACUXATF-ACZMJKKPSA-N Ser-Gln-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O FMDHKPRACUXATF-ACZMJKKPSA-N 0.000 description 2
- UOLGINIHBRIECN-FXQIFTODSA-N Ser-Glu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UOLGINIHBRIECN-FXQIFTODSA-N 0.000 description 2
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 2
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 2
- NLOAIFSWUUFQFR-CIUDSAMLSA-N Ser-Leu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O NLOAIFSWUUFQFR-CIUDSAMLSA-N 0.000 description 2
- FZEUTKVQGMVGHW-AVGNSLFASA-N Ser-Phe-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZEUTKVQGMVGHW-AVGNSLFASA-N 0.000 description 2
- HHJFMHQYEAAOBM-ZLUOBGJFSA-N Ser-Ser-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O HHJFMHQYEAAOBM-ZLUOBGJFSA-N 0.000 description 2
- PPCZVWHJWJFTFN-ZLUOBGJFSA-N Ser-Ser-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPCZVWHJWJFTFN-ZLUOBGJFSA-N 0.000 description 2
- VLMIUSLQONKLDV-HEIBUPTGSA-N Ser-Thr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VLMIUSLQONKLDV-HEIBUPTGSA-N 0.000 description 2
- BDMWLJLPPUCLNV-XGEHTFHBSA-N Ser-Thr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BDMWLJLPPUCLNV-XGEHTFHBSA-N 0.000 description 2
- PCMZJFMUYWIERL-ZKWXMUAHSA-N Ser-Val-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMZJFMUYWIERL-ZKWXMUAHSA-N 0.000 description 2
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- JEDIEMIJYSRUBB-FOHZUACHSA-N Thr-Asp-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O JEDIEMIJYSRUBB-FOHZUACHSA-N 0.000 description 2
- FHDLKMFZKRUQCE-HJGDQZAQSA-N Thr-Glu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FHDLKMFZKRUQCE-HJGDQZAQSA-N 0.000 description 2
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-N 0.000 description 2
- YDWLCDQXLCILCZ-BWAGICSOSA-N Thr-His-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YDWLCDQXLCILCZ-BWAGICSOSA-N 0.000 description 2
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 2
- BDGBHYCAZJPLHX-HJGDQZAQSA-N Thr-Lys-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O BDGBHYCAZJPLHX-HJGDQZAQSA-N 0.000 description 2
- HPQHHRLWSAMMKG-KATARQTJSA-N Thr-Lys-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)O)N)O HPQHHRLWSAMMKG-KATARQTJSA-N 0.000 description 2
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 2
- VUXIQSUQQYNLJP-XAVMHZPKSA-N Thr-Ser-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N)O VUXIQSUQQYNLJP-XAVMHZPKSA-N 0.000 description 2
- GRIUMVXCJDKVPI-IZPVPAKOSA-N Thr-Thr-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GRIUMVXCJDKVPI-IZPVPAKOSA-N 0.000 description 2
- BJJRNAVDQGREGC-HOUAVDHOSA-N Thr-Trp-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O BJJRNAVDQGREGC-HOUAVDHOSA-N 0.000 description 2
- LAIUAVGWZYTBKN-VHWLVUOQSA-N Trp-Asn-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(O)=O LAIUAVGWZYTBKN-VHWLVUOQSA-N 0.000 description 2
- SEXRBCGSZRCIPE-LYSGOOTNSA-N Trp-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O SEXRBCGSZRCIPE-LYSGOOTNSA-N 0.000 description 2
- NQJDICVXXIMMMB-XDTLVQLUSA-N Tyr-Glu-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O NQJDICVXXIMMMB-XDTLVQLUSA-N 0.000 description 2
- NMKJPMCEKQHRPD-IRXDYDNUSA-N Tyr-Gly-Tyr Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 NMKJPMCEKQHRPD-IRXDYDNUSA-N 0.000 description 2
- VBFVQTPETKJCQW-RPTUDFQQSA-N Tyr-Phe-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VBFVQTPETKJCQW-RPTUDFQQSA-N 0.000 description 2
- YKBUNNNRNZZUID-UFYCRDLUSA-N Tyr-Val-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YKBUNNNRNZZUID-UFYCRDLUSA-N 0.000 description 2
- XQVRMLRMTAGSFJ-QXEWZRGKSA-N Val-Asp-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XQVRMLRMTAGSFJ-QXEWZRGKSA-N 0.000 description 2
- BRPKEERLGYNCNC-NHCYSSNCSA-N Val-Glu-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N BRPKEERLGYNCNC-NHCYSSNCSA-N 0.000 description 2
- WFENBJPLZMPVAX-XVKPBYJWSA-N Val-Gly-Glu Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O WFENBJPLZMPVAX-XVKPBYJWSA-N 0.000 description 2
- AEMPCGRFEZTWIF-IHRRRGAJSA-N Val-Leu-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O AEMPCGRFEZTWIF-IHRRRGAJSA-N 0.000 description 2
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 2
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 2
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 2
- BZDGLJPROOOUOZ-XGEHTFHBSA-N Val-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N)O BZDGLJPROOOUOZ-XGEHTFHBSA-N 0.000 description 2
- UVHFONIHVHLDDQ-IFFSRLJSSA-N Val-Thr-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O UVHFONIHVHLDDQ-IFFSRLJSSA-N 0.000 description 2
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 2
- GUIYPEKUEMQBIK-JSGCOSHPSA-N Val-Tyr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(O)=O GUIYPEKUEMQBIK-JSGCOSHPSA-N 0.000 description 2
- RTJPAGFXOWEBAI-SRVKXCTJSA-N Val-Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RTJPAGFXOWEBAI-SRVKXCTJSA-N 0.000 description 2
- AOILQMZPNLUXCM-AVGNSLFASA-N Val-Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN AOILQMZPNLUXCM-AVGNSLFASA-N 0.000 description 2
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 2
- WHNSHJJNWNSTSU-BZSNNMDCSA-N Val-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 WHNSHJJNWNSTSU-BZSNNMDCSA-N 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 2
- 108010045023 alanyl-prolyl-tyrosine Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 229940064004 antiseptic throat preparations Drugs 0.000 description 2
- 108010008355 arginyl-glutamine Proteins 0.000 description 2
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 2
- 108010036533 arginylvaline Proteins 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000013320 baculovirus expression vector system Methods 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 229910001431 copper ion Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 108010078144 glutaminyl-glycine Proteins 0.000 description 2
- 108010080575 glutamyl-aspartyl-alanine Proteins 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 108010075431 glycyl-alanyl-phenylalanine Proteins 0.000 description 2
- 108010074027 glycyl-seryl-phenylalanine Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010010147 glycylglutamine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 108010087823 glycyltyrosine Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 230000007236 host immunity Effects 0.000 description 2
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 235000021056 liquid food Nutrition 0.000 description 2
- 108010003700 lysyl aspartic acid Proteins 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229940051866 mouthwash Drugs 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000013615 primer Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108010079317 prolyl-tyrosine Proteins 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 230000002685 pulmonary effect Effects 0.000 description 2
- 239000000985 reactive dye Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 2
- 235000012239 silicon dioxide Nutrition 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 108010015385 valyl-prolyl-proline Proteins 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- 101150005709 ARG4 gene Proteins 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- HZPSDHRYYIORKR-WHFBIAKZSA-N Asn-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O HZPSDHRYYIORKR-WHFBIAKZSA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- RXKJFZQQPQGTFL-UHFFFAOYSA-N Dihydroxyacetone Natural products OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- PMVSDNDAUGGCCE-TYYBGVCCSA-L Ferrous fumarate Chemical compound [Fe+2].[O-]C(=O)\C=C\C([O-])=O PMVSDNDAUGGCCE-TYYBGVCCSA-L 0.000 description 1
- 206010051283 Fluid imbalance Diseases 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- AKJRHDMTEJXTPV-ACZMJKKPSA-N Glu-Asn-Ala Chemical compound C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AKJRHDMTEJXTPV-ACZMJKKPSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 208000018565 Hemochromatosis Diseases 0.000 description 1
- 206010022971 Iron Deficiencies Diseases 0.000 description 1
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 101150077375 LF gene Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- OKIZCWYLBDKLSU-UHFFFAOYSA-M N,N,N-Trimethylmethanaminium chloride Chemical compound [Cl-].C[N+](C)(C)C OKIZCWYLBDKLSU-UHFFFAOYSA-M 0.000 description 1
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 208000007027 Oral Candidiasis Diseases 0.000 description 1
- 206010048685 Oral infection Diseases 0.000 description 1
- 102100033118 Phosphatidate cytidylyltransferase 1 Human genes 0.000 description 1
- 101710178747 Phosphatidate cytidylyltransferase 1 Proteins 0.000 description 1
- 208000035109 Pneumococcal Infections Diseases 0.000 description 1
- 101710182846 Polyhedrin Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 206010041925 Staphylococcal infections Diseases 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 206010061372 Streptococcal infection Diseases 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 101710136739 Teichoic acid poly(glycerol phosphate) polymerase Proteins 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010046914 Vaginal infection Diseases 0.000 description 1
- 101100004044 Vigna radiata var. radiata AUX22B gene Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 201000007096 Vulvovaginal Candidiasis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- CIUQDSCDWFSTQR-UHFFFAOYSA-N [C]1=CC=CC=C1 Chemical class [C]1=CC=CC=C1 CIUQDSCDWFSTQR-UHFFFAOYSA-N 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- SNAAJJQQZSMGQD-UHFFFAOYSA-N aluminum magnesium Chemical compound [Mg].[Al] SNAAJJQQZSMGQD-UHFFFAOYSA-N 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 108010038047 apolactoferrin Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000003103 bodily secretion Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 201000007717 corneal ulcer Diseases 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940120503 dihydroxyacetone Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 229910001447 ferric ion Inorganic materials 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 208000024386 fungal infectious disease Diseases 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000009215 host defense mechanism Effects 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000000091 immunopotentiator Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000002997 ophthalmic solution Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000250 revascularization Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000021055 solid food Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000011272 standard treatment Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 235000002374 tyrosine Nutrition 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/18—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from yeasts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3481—Organic compounds containing oxygen
- A23L3/3508—Organic compounds containing oxygen containing carboxyl groups
- A23L3/3517—Carboxylic acid esters
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/19—Dairy proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates generally to the glycoprotein lactoferrin, its production, purification, and uses. BACKGROUND OF THE INVENTION
- Lactoferrin plays an important role in iron transport and utilization in humans.
- a glycoprotein containing about 6% sugar and having a total molecular weight of about 80,000 daltons, human lactoferrin is capable of binding two ferric ions with high affinity.
- lactoferrin binds iron tightly, the binding is reversible so that the metal is available upon demand to cells with a need for this essential element.
- the metal is captured by the side chains of specific amino acids: two tyrosines, one histidine and one aspartate which in combination form a cleft in the surface of the protein. That portion of the protein which contains the aforesaid four amino acids and forms the cleft is termed the “iron-binding domain.”
- Each natural lactoferrin molecule has two iron-binding domains.
- Human milk is high in lactoferrin content.
- the high degree of iron absorption from human milk is manifested in a low incidence of iron deficiency anemia among breast fed infants compared to infants fed with cow's milk.
- lactoferrin is a key protein for healthy development of infants.
- Lactoferrin also plays an important role in cell-mediated host immunity. It is present in high concentrations in all bodily secretions, such as tears, sweat, and ciliated respiratory mucous. Because it sequesters iron, lactoferrin can neutralize pathogenic microorganisms by preventing them from obtaining necessary iron at the site of entry, thereby preventing the spread of infection.
- iron is an essential material in humans, excess iron in the body can induce pathological conditions as well.
- Chronic iron overload known as hemosiderosis, is characterized by a greater than normal local or generalized deposition of iron within certain body tissues. Lactoferrin helps to manage the balance of free iron in the body to prevent occurrence of such pathological states in healthy individuals.
- the present invention provides for the cloning and expression of human lactoferrin using recombinant DNA techniques.
- the lactoferrin of the present invention is free of naturally occurring contaminants, e.g., proteins and viruses, that would be detrimental to the recipient.
- a gene comprising a DNA molecule encoding human lactoferrin protein. More particularly, the DNA molecule comprises the nucleotide sequence (Seq. ID No. 1) and the protein comprises the amino acid sequence (Seq. ID No. 2) as substantially depicted in FIG. 3.
- An expression system is provided for expressing the gene encoding the protein. Preferably, the expression system is a plasmid.
- a host cell line transformed with the gene of the present invention i.e., the gene encoding human lactoferrin.
- the host cell organism is a eukaryotic cell.
- a method of producing human lactoferrin comprising the steps of (a) isolating a gene encoding human lactoferrin; (b) transforming a host cell with the gene; (c) culturing the transformed cells which express the gene product; and (d) collecting lactoferrin from the cells.
- a chromatography method for purifying lactoferrin protein, and other proteins comprising the steps of (a) contacting a substance with a first adsorbent to obtain adsorbed and non-absorbed fractions; (b) eluting the adsorbed fraction with an eluant; and (c) contacting the adsorbed fraction with a second adsorbent, wherein the improvement comprises equilibrating the second adsorbent with the eluant followed by contacting the eluate containing the adsorbed fraction with the second adsorbent.
- a method for inhibiting microbial growth in a mammal comprising topically or systemically applying to a subject a therapeutically effective amount of lactoferrin having less than about 25% metal loading; a method for inhibiting iron deficiency in a mammal comprising orally administering a therapeutically effective amount of lactoferrin having at least about 35% iron loading; a nutritional supplement comprising an iron-loaded human lactoferrin having at least about 35% metal loading and a nutritionally acceptable carrier or adjuvant; a topical antiseptic comprising a therapeutically effective amount of lactoferrin having less than about 25% metal loading and a pharmaceutically acceptable carrier or diluent; and a method for inhibiting food spoilage comprising adding to the food an effective amount of lactoferrin having less than about 25% metal loading.
- FIG. 1 is a schematic diagram showing the chromatography method of the invention.
- FIG. 2 is a flow chart showing the purification of lactoferrin.
- FIGS. 3 a - 3 d show the nucleotide sequence (Seq. ID No. 1) and the deduced amino acid sequence (Seq. ID No. 2) of the gene encoding human lactoferrin.
- FIG. 4 is an agarose gel analysis of amplified cDNA coding human lactoferrin.
- FIG. 5 is a map of the pUC118 plasmid.
- FIG. 6 is a map of the pHIL-D1 plasmid.
- FIG. 7 is a map of the pPIC9 plasmid.
- FIG. 8 is a restriction fragment map of the cDNA lactoferrin gene.
- FIG. 9 is an SDS-PAGE analysis of recombinantly expressed lactoferrin according to the present invention.
- FIG. 10 is a Western blot analysis of the recombinantly expressed lactoferrin according to the present invention.
- Lactoferrin is produced in accordance with the present invention using recombinant DNA technology to produce genetically modified DNA that expresses lactoferrin.
- the recombinant DNA technology described herein is standard technology in the art, such as described by Maniatis, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Press, 1989, Chapter 14, which disclosure is hereby incorporated by reference. That is, a polypeptide containing the primary structural conformation of the naturally occurring human lactoferrin protein, having similar biological (i.e. physical) properties is produced.
- the DNA encoding lactoferrin is preferably from a cDNA library derived from human RNA and ligated to an appropriate expression vector according to standard techniques in the art, such as those disclosed in Davis, et al., Basic Methods in Molecular Biology , Elsevier Science Publishing Co. (1986), which disclosure is hereby incorporated by reference.
- the RNA is isolated from the human mammary gland and the preferred vector is phage ⁇ gt11.
- the cDNA library is screened for positive (DNA carrying the lactoferrin gene) clones using conventional techniques in the art, such as disclosed by Davis, et al., supra, and Rado, et al., Blood, 70:4, 989-993 (October, 1987), which disclosure is hereby incorporated by reference.
- the cDNA is hybridized to a radiolabeled oligonucleotide probe and the positive clones identified by auto radiography.
- positive clones are identified using lactoferrin antisera, and an appropriate development system, such as an avidin/biotin reaction system. Large numbers of positive clones are then generated by infecting an appropriate bacterial host, such as E.
- DNA is then isolated from the clones.
- the cDNA encoding lactoferrin is thereafter cut with an appropriate restriction endonuclease such as EcoRI.
- the cut DNA encoding lactoferrin is separated by chromatography.
- the separated cDNA is further sub-cloned into another vector, such as the pGEM-4 plasmid, and the inserted cDNA again excised and separated.
- yeast Pichia pastoris with an appropriate expression vector such as those driven by the alcohol oxidase promoter and disclosed in Tschopp, Nucleic Acid Research , Vol. 15, pp. 3859-3876, (1987).
- Other useful hosts include: (i) the yeast Hansenula polymorpha with an appropriate expression vector driven by strong promoters such as methanol oxidase (MOX), dihydroxyacetone synthetase (DAS), or formate dehydrogenase (FMDH) as disclosed in Gellisen, Biotech. Adv ., Vol 10, pp.
- MOX methanol oxidase
- DAS dihydroxyacetone synthetase
- FMDH formate dehydrogenase
- Insertion of the cDNA and expression of the human lactoferrin are carried out using standard recombinant techniques that are readily apparent to one skilled in the art, such as described in Rothstein, Methods in Enzymology, 101, 202-210 (1983); and Tschopp, et al., Bio/Technology, 5, 1305-1308 (1987), which disclosures are hereby incorporated by reference.
- the gene is amplified from a human mammary gland library using the polymerase chain reaction (PCR) technique.
- PCR polymerase chain reaction
- amplification of the insert encoding the lactoferrin gene is achieved by using two synthetic oligonucleotide probes corresponding to the amino acid sequence residues 1 to 9 of the amino terminus and residues 2070 to 2079 of the carboxy terminus.
- the amplified insert is cut out with restriction enzymes BamH I and Xba I, then purified, and ligated into an appropriate vector, preferably pBlueScripKS+, which is digested with the same restriction enzymes.
- the lactoferrin gene is further subcloned into pUC118 vector, cut with Hind III and Sst I to produce a plasmid designated pUC118-LF, containing the coding region for the mature lactoferrin protein.
- Secretion of the lactoferrin protein may be further enhanced by further manipulations of plasmid pUC118-LF. For example, the following two methods are provided for purposes of illustration.
- the first method is the addition of a signal sequence to the 5′ end of the clone.
- the preferred signal sequence is that described by Powell, et al., Nucleic Acids Research, 18, 4013 (1990), which disclosure is hereby incorporated by reference.
- pUC118-LF modified with the aforementioned signal sequence is defined as pUC118-LFS.
- the lactofernin gene, together with the signal sequence is cut from pUC118-LFS and then ligated to pHIL-D1 to produce pHIL-D1-LFS.
- the second, and more preferable, method employs the alpha mating factor pre-pro secretion signal.
- the mature lactofernin gene is cut from plasmid pUC118-LF and ligated to plasmid pPIC9 which had been previously ligated with an alpha mating factor pre-pro secretion signal.
- An additional embodiment of the present invention relates to fragments of both the lactoferrin gene and lactoferrin.
- the active sites of the lactoferrin protein are the iron-binding domains. Protein sequences which contain one or more of the iron-binding domains of the lactoferrin protein sequester iron and, therefore, are useful in the antiseptic, dietary supplement, and food-spoilage retardant embodiments of the present invention.
- the present invention contemplates a (DNA) fragment of the DNA molecule encoding human lactoferrin. The fragment encodes a portion of the human lactoferrin protein containing at least one of the iron-binding domains.
- a DNA fragment of the lactoferrin gene encoding only one iron-binding domain of human lactoferrin can be obtained through a specific design of oligonucleotides for PCR amplification or through an antibody probe screening of a cDNA library, which procedures are readily apparent to one skilled in the art. These fragments are also useful, e.g., as intermediates in the synthesis of the full-length gene and protein.
- a cell-free culture media containing the expressed lactoferrin protein is passed through a filter that retains material having a molecular weight greater than about 10,000 daltons and then sterilizing the retained protein.
- the material retained by the filter is subjected to a two-step affinity chromatography process.
- the affinity ligand is the reactive dye Cibacron blue F3G-A (color index (C.I.) 61211, ⁇ max 605(374)mn) as disclosed by Bezwoda, et al., Clin. Chim.
- the adsorbed material from the CPG or silicic acid is further chromatographed in a third step using one of the following chromatography techniques before final filtration and sterilization: T-Gel chromatography; immobilized metal-ion affinity chromatography (IMAC) using a metal ion capable of forming a complex with lactoferrin, such as nickel or copper, which can be coupled with an imminodiacetic acid-epoxy activated gel (IDA Me(II)) as described by Sulkowski, Frontiers in Bioprocessing , Sidkar et al., Ed., 343-353 (1990), which disclosure is hereby incorporated by reference; or chromatography with the ligand phenyl glycidyl ether, which can be coupled to a cross-linked agarose gel as described by Janson, et al., Protein Purification Principles High Resolution Methods and Applications, VSH Publishers, New York (1989), which disclosure is hereby incorporated by reference.
- IMAC immobilized metal-
- the improved chromatography process of the present invention is useful in purifying proteins, such as lactoferrin produced in accordance with the present invention.
- crude fermentation broth contained in tank 1 passes to permeable membrane 3 , which retains material having a molecular weight greater than 10,000 daltons and passes an ultra filtrate containing water, salts, and low molecular-weight proteins.
- the retained material is washed with a buffer and further concentrated.
- the washed material is then applied to chromatography column 5 containing an adsorbent that has been equilibrated with the buffer used to wash the filtered material while valve 7 is open and valve 9 is closed. After non-adsorbed material is discharged through valve 7 , valve 7 is closed and valve 9 opened.
- Adsorbed material is then eluted, and the eluate passed directly to the second column 11 , containing an adsorbent previously equilibrated with the eluant used to elute the adsorbed material.
- Use of the same medium to elute material from the adsorbent in column 5 and equilibrate the adsorbent column 11 avoids the need for timely and involved medium exchange procedures between the two adsorption steps. Passage of the adsorbed material through column 11 occurs while valve 13 is open and valve 15 is closed.
- Eluting adsorbed material from column 11 occurs while valve 13 is closed and valve 15 is open, thereby passing eluate from column 11 directly to a filter (not shown) capable of retaining material having a molecular weight of at least 10,000 daltons.
- a filter capable of retaining material having a molecular weight of at least 10,000 daltons.
- the lactoferrin of the present invention having either no metal loading (iron-free lactoferin, apolactoferrin) or a low metal loading, preferably less than 25%, more preferably less than 20%, most preferably less than 10% of the metal-binding sites occupied, by virtue of being capable of sequestering a significant amount of iron, is useful in applications to individuals where the removal of iron or other transition metals from the individual can have beneficial effects, such as in cosmetics, personal hygiene products, such as feminine douches and mouthwashes, medical and surgical devices and products, topical antiseptics, ophthalmic solutions, oral and intravenous antibiotics, immunopotentiators, antioxidant, and anti-inflammatory and anti-tumor agents.
- Lactoferrin can be used as an antiseptic in accordance with the present invention either alone or in the form of a powder, solution, ointment, aerosol spray, or cream to any part of the subject as an aid in the prevention or treatment of microbial infections.
- lactoferrin inhibits the growth of microbes, such as bacteria
- Preferable antiseptics of the present invention include lactoferrin either alone or compounded with carriers such as saline, silica, talcum, stearic acid, its magnesium or calcium salt, polyethyleneglycol, and fatty emulsions and suspensions that will be readily apparent to the skilled artisan.
- the lactoferrin is preferably present in the antiseptic based on 1 ml of the carrier at 0.1-2 mg, preferably 0.2-2 mg.
- An effective amount of the lactoferrin varies depending on the individual treated, severity of infection, if any, and the area to which administration is contemplated.
- a twice-daily administration of 0.1-2 mg, more preferably 0.5-2 mg, most preferably 1 mg, of lactofernin per 1 square centimeter effected area is contemplated more preferably 0.1 square centimeter.
- Lactoferrin can be used as an antiseptic in accordance with the present invention to treat accidental scratches or burns.
- lactoferrin provides added protection against sexually transmitted infections when compounded into any device used during sexual activities by either males or females.
- lactoferrin is added into lubricant to cover condoms at the concentration of 25-50 mg per application.
- Lactoferrin can be used as antiseptic in accordance with the present invention to impregnate any surgical tools, materials or protective clothing that is used by health care personnel.
- surgical gloves, masks or linens are covered with a 0.1-2.0% by weight solution of lactoferrin, preferably 0.1-1% by weight.
- the solution can be applied by a spray, conveniently provided in pressurized aerosol cans or pump bottles.
- Lactoferrin can be useful in the treatment and prevention of opportunistic bacterial, viral, and fungal infections.
- Opportunistic infections are caused by normally non-pathogenic organisms in patients whose host defense mechanisms have been compromised. By sequestering iron, lactoferrin inhibits the growth of these organisms, making them more susceptible to antibiotic therapy.
- treatment can involve one or more types of systemic (oral, nasal, intravenous, etc.) or topical administration. Examples of such infections include pneumonia, acquired immune deficiency syndrome (AIDS), candidiasis, diarrhea, and neonatal sepsis.
- AIDS acquired immune deficiency syndrome
- lactoferrin In treating pneumonia, for example pneumonia caused by Streptococcus pneumoniae , antibiotics have minimal impact on mortality during the first five days of illness. By sequestering iron, lactoferrin can inhibit the growth of pneumococcals, and make them more susceptible to antibiotic therapy.
- administration by oral and intravenous routes is contemplated, a simple delivery system of lactoferrin by inhalation involving topical administration to pulmonary membranes is most preferred.
- treatment will involve administration three to four times daily of an aqueous solution of lactoferrin in an amount of 100-200 mg per dose for a period of time of 7 to 10 days by inhalation using a known inhaler.
- a particular cause of opportunistic infections is the lowered host immunity caused by AIDS.
- a pulmonary infection such as Pneumocistis carinii
- the treatment will involve administration of lactoferrin by inhalation four times daily in an amount of 100-200 mg per dose for a period of time of 7 to 10 days.
- Kaposi's sarcoma treatment will involve topical administration of lactoferrin in an ointment, twice daily, in an amount of 50-100 mg per dose for three to four weeks.
- Fungi infections depending on the type and location, are treated orally, by intravenous injection, or topical administration.
- infections such as vaginal candidiasis
- vaginal wash vaginal wash twice daily in an amount of 100-200 mg per dose.
- Diarrhea while not usually life threatening, can be dangerous, particularly in infants, because of the potential for fluid imbalance.
- lactoferrin can inhibit the growth of pathogens in the gastrointestinal tract.
- Treatment of diarrhea will involve oral administration of lactoferin twice daily at an amount of 100-200 mg per dose for a period of time of 7 to 10 days.
- the treatment of ulcers caused by Helicobacter pylori is also facilitated by the use of lactoferrin. Lactoferrin will not only prevent utilization of iron by the bacteria by sequestering excessive iron from food, it will also deliver the iron to the small intestine where it is recognized by receptors specific to lactoferrin.
- Treatment will involve oral administration of lactoferrin twice daily in an amount of 200-400 mg per dose for a period of time of 7 to 10 days.
- Neonatal sepsis which can coincide with low production levels of lactoferrin in newborn infants, is also subject to treatment by lactoferrin in accordance with the present invention, and particularly in combination with current antibacterial therapy.
- Treatment will include intravenous administration of lactoferrin twice daily in an amount of 100-200 mg per dose for a period of time of 7 to 10 days.
- the plasma level of lactoferrin increases 10 to 20 times the normal amount, the body responding to injury by secreting a powerful antimicrobial agent-lactoferrin.
- Topical administration of lactoferrin to burn patients, by sequestering iron, will prevent development of surface infection.
- Treatment will involve topical administration of lactofernin in an ointment, cream, or other topical vehicle twice daily in an amount of 50-100 mg per dose for a period of time of three to four weeks.
- Chronic iron overload known as hemosiderosis, is characterized by greater than normal iron levels in certain body tissues. When associated with tissue injury, the condition is known as hemochromatosis.
- Lactoferrin as the natural chelating agent for iron, offers a viable treatment for such disorders.
- Preferable treatment involves intravenous or subcutaneous doses of lactoferrin once daily at an amount of 300-500 mg.
- Lactofernin can also be used to sequester iron implicated in heart disease. By sequestering iron that promotes the oxidation of lipids, which when oxidized can clog arteries, lactoferrin can aid in reducing heart attacks.
- the prophylactic treatment involves intravenous administration of lactoferrin twice weekly in an amount of 200-500 mg. The treatment is contemplated for high risk patients having high levels of cholesterol. Ischemic heart disease remains the most important cause of morbidity and mortality in the U.S. Over the last decade acute revascularization with thrombolytic drugs has emerged as the standard treatment for patients with acute myocardial infraction. Considerable evidence has emerged over the last decade which indicates that iron may play a key role in pathogenesis of reperfusion injury in the heart.
- Lactoferrin can sequester large amounts of iron that tend to be released following heart attacks, which thus reduces the amount of iron available for reacting with oxygen to generate free radicals, which cause damage to muscle fibers and cell walls.
- Preferable treatment involves intravenous administration of lactoferrin immediately after heart attack in an amount of 500-1,000 mg.
- the treatment of a pneumococcal, streptococcal or staphylococcal infection following trauma of the cornea is also facilitated by lactoferrin. These infections are usually primary causes of corneal ulcers.
- the treatment will involve topical administration of lactoferrin in an ointment, eye drops or other topical vehicle twice daily in an amount of 10-20 mg per application.
- Lactoferrin can also be utilized to sequester iron from contact lenses having an application in antiseptic treatment of lenses between wearing times.
- the effective solution will consist of 0.1-1.0% of lactoferrin in water.
- the treatment of tumors, such as brain tumors, can also be facilitated by the use of lactoferrin.
- Newly developed catherization procedures permit the delivery of lactoferrin directly to a blood supply aorta of a tumor, such as a brain tumor, which, by reducing the iron necessary for metabolism of the tumor cells, can prevent the growth of the tumor.
- Treatment will involve weekly administration of lactoferrin through a microcatheter in an amount of 1-2 grams per dose for a period of time of three to six months.
- Lactoferrin can also be used as an adjuvant in vaccination.
- Subunit antigens and peptides made by recombinant DNA technology are not very immunogenic, making their use as vaccines contingent upon the availability of adjuvants that are safe for use in humans and are able to augment sufficiently the immunogenicity of these molecules. Because it modulates a number of immunological responses including myeloid differentiation, modulation of macrophage-mediated cytotoxicity, and regulation of the primary antibody response, lactoferrin can be used as such an adjuvant.
- the lactoferrin is systemically administered at 100-200 mg per vaccination. Lactoferrin administration before, during, or after vaccination is contemplated based on the specific antigen used for the vaccine.
- the nutritional supplement of the present invention contains an effective amount of lactoferrin loaded with iron, either alone or in combination with one or more nutritionally acceptable carriers or adjuvants.
- Preferred nutritional supplements include tablets, gelatin capsules, or liquids containing the lactoferrin together with adjuvants or diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine; binders, such as magnesium aluminum silicate, starch, paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone; disintegrants, such as starches, agar, alginic acid or its sodium salt, and effervescent mixtures; as well as absorbents, colorants, flavors and sweeteners.
- adjuvants or diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and
- the iron-loaded lactoferrin can be added to foods such as baby formula, cereal, and ice cream to enhance the nutritional value of the food.
- the preferred amount of iron-loaded lactofernin in the supplement based on the weight of 1 g of the supplement is 5-50 mg, more preferably 20-30 mg, and most preferably 25 mg.
- An effective daily amount of based on the individual, iron-loaded lactoferrin varies, from about 10-30 mg, preferably 20-30 mg, and more preferably 25 mg. Loading lactoferrin with iron is accomplished by simple titration with, e.g., ferrous ammonium in the presence of bicarbonate, according to methods that will be readily apparent to the skilled artisan.
- Preferred loading is such that at least about 35%, more preferably at least about 50%, and most preferably at least about 70%, of the metal binding sites are iron bound.
- Lactoferrin can be applied to food (either solid or liquid) to retard spoilage in accordance with the present invention either alone or compounded with any of the aforesaid nutritionally acceptable carriers or diluents. By sequestering iron, and thereby suppressing its catalytic activity, the lactoferrin reduces the iron available for either microbial multiplication or the production of potentially cell-damaging free-radicals that are formed in iron catalyzed reactions from hydrogen peroxide or superoxide.
- the lactoferrin is particularly useful in inhibiting rancidity in meat, which is iron-dependent lipid peroxidation.
- the lactoferrin can be added directly to the liquid or used to coat filters through which the liquid food passes during processing.
- An effective amount of the lactoferrin for retarding spoilage varies depending on the type and amount of food contemplated.
- the amount of lactoferrin applied to food in accordance with the present invention varies from 0.1-1 mg/ml of food with which it is mixed, or based on the surface area of the filter or solid food to which it is applied from 0.1-1 mg/cm 2 .
- the preferred amount of lactoferrin compounded with a carrier in a food additive for retarding spoilage varies based on 1 ml of the carrier from 0.1-2 mg, preferably 0.2-2 mg.
- the antiseptic, dietary supplement, and food-spoilage retardant of the present invention can be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
- adjuvants such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating osmotic pressure and/or buffers.
- they may also contain other therapeutically valuable substances.
- Said compositions are prepared according to conventional mixing, granulating, and coating methods.
- lactoferrin contemplated for use in accordance with the present invention is preferably of human origin, more preferably via DNA recombinant means, but other lactoferrins, such as natural bovine, goat and porcine lactoferrin, isolated and purified using methods applicable to natural human lactoferrin, are contemplated.
- Human lactoferrin is obtained from a genetically altered organism. Using breast tissue excised during the mastectomy of a woman during the eighth month of pregnancy, a human mammary gland genomic library is prepared according to the procedure of Gubler, et al., Gen., 40, 1-8 (1983), which disclosure is hereby incorporated by reference.
- a human mammary gland genomic library of cDNA ligated to ⁇ gt11 is available from Clontech, Calif.
- the library is transferred onto agar plates containing a high density of E. coli Y 1090 (Clontech, Calif.) (5 ⁇ 10 4 plaques per 90 mm plate or 1.4 ⁇ 10 5 plaques per 150 mm plate). The plates are allowed to stand for 3.5 hours at 42° C. to obtain a lytic growth of the phage.
- the plates are thereafter overlaid with nitrocellulose filters (Schleiher and Schnell Inc., Wouburn, Mass., under no. BA 85 NC) and heated in an incubator at 37° C. for 3.5 hours
- Positive clones i.e., containing the cDNA are identified on the membranes using rabbit antibody to natural human lactoferrin purified in accordance with Example 9 described hereafter. Nitrocellulose filters are removed from the plates after plaque transferral, and coated with the antibody purified as described under Example 9, which hybridizes with positive plaques. Following removal of excess antibody, positive plaques are developed by first applying an anti-rabbit IgG conjugated with biotin (Sigma Chemical Co., St. Louis, Mo.), and then, following removal of excess biotin conjugate, applying avidin conjugated with horse radish peroxidase (Sigma Chemical Co., St. Louis, Mo.). Finally, the positive plaques are identified in the reaction catalyzed by horse radish peroxidase using as an enzyme substrate 4chloro-1-Naphtol.
- the positive plaques are then used to infect E. coli Y 1090 to produce large amounts of phage in accordance with procedures set forth in Davis, et. al. (1986), supra.
- the resulting bacteriophage is purified using 10% polyethylene glycol and DNA is isolated from the phage according to the procedures disclosed in Kislow, N. A. R., 14, 6767 (1986), which disclosure is hereby incorporated by reference. Following the procedures in Davis, et al.
- the cDNA insert encoding lactoferrin is sub-cloned as follows: the cDNA insert is cut out from the phage DNA using EcoR I and purified using a high resolution ion-exchange chromatography column (GEN-PAKTM Fax, Millipore Corporation, Waters Chromatography Division, Milford, Mass.). The thus purified cDNA insert is ligated using T4 DNA ligase into plasmid pGEM-4 (Promega, Madison, Wis.) as described by Yanish-Perron et al., Gen., 33, 103-109 (1985), which disclosure is hereby incorporated by reference that has been cut using EcoR I using standard techniques.
- the plasmid containing the insert is then transferred into E. coli JM109 (Promega, Madison, Wis.) as described by Hanahan, J. Mol. Blot, 166, 557 (1983), which disclosure is hereby incorporated by reference.
- the bacteria are transferred to agar plates containing ampicillin and the positive colonies grown.
- the plasmid is then isolated and the cDNA insert is cut from the plasmid using EcoR I and purified by ion exchange chromatography as described above.
- the cDNA insert is then ligated into the Pichia pastoris expression vector pAO804, so as to be flanked by the 5′ and 3′ regulatory sequences of the methanol-induced alcohol oxidase gene (AOX1) of P. pastoris in accordance with the procedures described by Sreekrishna, et al., Biochemistry, 28, 4117-4125 (1989); and Rothstein, Methods in Enzymology, 101, 202-210 (1983), which disclosures are hereby incorporated by reference.
- the resulting vector is then transformed into Pichia pastoris GTS115 (His4) by the method of spheroplast as described by Creeg, et al., Mol. Cell.
- the selected transformant cells are grown in 10 ml buffered minimal glycerol-complex medium at 30° C. with shaking for three days.
- the saturated culture is centrifuged for 10 minutes at 3000 G.
- the cell pellet is resuspended with 2 ml of buffered minimal methanol-complex medium and returned to the 30° C. shaker for another three days.
- Cells are pelleted by centrifugation and both the pellet and supernatant are analyzed for the presence of lactoferin.
- Hagenson et al. Enzyme Microb.
- Human lactoferrin is purified using affinity chromatography in which the affinity ligand is the reactive dye Cibacron blue F3G-A.
- the sterilized material obtained in Example 2 is adjusted to a pH of 7.5 and a final concentration of sodium chloride of 0.5 M.
- This material is then applied onto a column (5 cm ⁇ 35 cm) packed with cross-linked agarose coupled to the dye (Pharmacia Fine Chemicals, Uppsala, Sweden, BLUE SEPHAROSETM CL-6B) and previously equilibrated with 50 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) buffer (pH 7.5) containing 0.5 M sodium chloride.
- HEPHAROSETM CL-6B cross-linked agarose coupled to the dye
- Adsorption is performed at a flow rate of 1 ml/min followed by washing the column with 2 bed volumes of the same HEPES buffer. The non-adsorbed fraction is discarded and the adsorbed fraction containing lactoferrin is eluted from the column bed using 2 bed volumes of 50 mM HEPES buffer (pH 7.5) containing 1 M sodium chloride.
- Human lactoferrin is purified using control pore glass (CPG) chromatography.
- CPG control pore glass
- the eluate from Example 3 is applied onto a column (1.2 cm ⁇ 10 cm) packed with CPG beads (CPG 00350, ElectroNucleonics, Fairfield, N.J.) and previously equilibrated with 50 mM HEPES buffer (pH 7.5) containing 1 M sodium chloride.
- Adsorption is performed at a flow rate of 1 ml/min followed by washing the column with 2 bed volumes of the same buffer.
- the non-adsorbed fraction is discarded, and the adsorbed fraction is eluted with 2 bed volumes of 0.25 M tetramethylammonium chloride (TMAC, pH 7.5).
- TMAC tetramethylammonium chloride
- the eluate is filtered on a membrane capable of excluding material having a molecular weight greater than 10,000 daltons (AMICONTM YM 10).
- the filtered material is then sterilized (0.22 micron GELMANTM filter) and frozen at ⁇ 200C.
- Human lactoferrin is purified using immobilized metal ion affinity chromatography (IMAC).
- IMAC immobilized metal ion affinity chromatography
- An imminodiacetic acid-epoxy activated gel (Pharmacia Fine Chemicals, Uppsala, Sweden, CHELATING SEPHAROSETM 6B) is washed with water and equilibrated with 0.1 M sodium acetate buffer (pH 4.0) containing 1 M sodium chloride.
- the gel is then packed into a chromatographic column (1.2 cm ⁇ 10 cm) and saturated with 4 bed volumes of the same sodium acetate buffer further containing 5 mg/ml of nickel chloride. Excess metal is washed from the column with the sodium acetate buffer, and the gel is equilibrated with 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 2 mM imidazole.
- Example 4 The product of Example 4 is mixed with HEPES, sodium chloride, and imidazole to obtain a pH of 7.0, 20 mM HEPES, 1 M sodium chloride, and 2 mM imidazole.
- the mixture is applied onto the column at a flow rate of about 1 ml/min followed by,washing the gel with 2 bed volumes of 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 2 mM imidazole.
- the non-adsorbed fraction is discarded, and the adsorbed fraction containing lactoferrin is eluted using 2 bed volumes of 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 20 mM imidazole.
- Human lactoferrin is purified using immobilized metal ion affinity chromatography (IMAC).
- IMAC immobilized metal ion affinity chromatography
- An imminodiacetic acid-epoxy activated gel (Pharmacia Fine Chemicals, Uppsala, Sweden, CHELATING SEPHAROSETM 6B) is washed with water and equilibrated with 0.1 M sodium acetate buffer (pH 4.0) containing 1 M sodium chloride.
- the gel is then packed into a chromatographic column (1.2 cm ⁇ 10 cm) and saturated with 4 bed volumes of the same sodium acetate buffer further containing 5 mg/ml of copper sulfate.
- Excess metal is washed from the column with the sodium acetate buffer, and the gel is equilibrated with 50 mM TRIS-HCL buffer (pH 8.0) containing 1 M sodium chloride.
- T-gel adsorbent is prepared according to the procedure described by Porath, et al., Methods in Enzymology, 44, 19-45 (1976), which disclosure is hereby incorporated by reference, and packed into a column (1.2 cm ⁇ 10 cm).
- the final product of Example 4 is adjusted to a pH of 7.5 and a final concentration as follows: 50 mM PIPES buffer (piperazine-N,N′-bis[2-ethanesulfonicacid] and 1,4-piperazinediethanesulfonicacid) buffer and 0.7 M ammonium sulfate.
- the adjusted material is applied on the column that has been previously equilibrated to 50 mM PIPES buffer (pH 7.5) containing 0.7 M ammonium sulfate with a flow rate of about 1 ml/min.
- the non-adsorbed fraction containing lactoferrin is adjusted to a concentration of 0.1 M ammonium sulfate and then applied to an identical T-gel column previously equilibrated to 50 mM PIPES buffer (pH 7.5) containing 1.0 M ammonium sulfate.
- the column is then washed with 7-8 bed volumes of 50 mM PIPES buffer (pH 7.5) containing 1.0 M ammonium sulfate, with lactoferrin being present in the non-adsorbed fraction.
- Human lactoferrin is purified using hydrophobic interaction chromatography on a cross-linked agarose gel coupled to phenyl glycidyl ether (PHENYL SEPHAROSETM CL-4B, Pharmacia Fine Chemicals, Uppsala, Sweden). The gel is packed into a column and equilibrated to 50 mM PIPES buffer (pH 7.0) containing 1 M ammonium sulfate. The product of Example 4 is adjusted to the equilibrating buffer and applied onto the column at a flow rate of 1 ml/min. The non-adsorbed fraction is discarded and the adsorbed fraction containing lactoferrin is eluted using 2 bed volumes of 50 mM PIPES buffer (pH 7.0).
- Anti-lactoferrin serum is purified by affinity chromatography.
- the adsorbent substrate for affinity chromatography is prepared by cyanogen bromide activation as described by Axen et al., Nature, 214, 1302-1304 (1967), which disclosure is hereby incorporated by reference.
- the substrate (Pharmacia Fine Chemicals, Uppsala, Sweden, CNBR-SEPHAROSETM 4B) is coupled to human lactoferrin, which acts as the affinity ligand, as follows. One gram of substrate is swelled with 1 mM HCl and washed with the same solvent on a sintered glass filter. Ten mg of natural human lactoferin (Sigma Chemical Co., St.
- Adsorbed material containing the purified protein is eluted with 2 bed volumes of 0.2 M glycine buffer (pH 2.0) containing 0.5 M of sodium chloride. The eluate is neutralized with 0.1N NaOH to obtain pH 7.5 and then sterilized (0.22 micron GELMANTM filter) and frozen at ⁇ 20° C.
- the human lactoferrin gene is cloned by PCR amplification from a human mammary gland library in ⁇ gt11.
- Phage DNA is prepared from the library by using standard procedures described in Maniatis (1989), supra.
- Oligonucleotide primers are prepared to the 5′- and 3′-ends of the gene based on the sequence published by Powell, et al. (1990), supra.
- the 5′-oligo is designed to begin at the coding sequence for the first glycine of the mature lactoferrin protein as defined by Powell, et al. (1990), supra, and the 3′-oligo included the lactoferrin stop codon and 21 nucleotides beyond.
- oligonucleotides are prepared on an Applied Biosystems DNA synthesizer Model 380A and were obtained from Roswell Park Cancer Institute, Buffalo, N.Y. Each oligo also created a restriction endonuclease recognition site, the 5′-oligo contained a BamH I site and the 3′-oligo an Xba I site. The sequences of these oligonucleotides are given below:
- PCR amplification of the cDNA library using these oligonucleotide primers is performed with a temperature cycler (Genetic Thermal Cycler, Precision Scientific), utilizing Tag polymerase. Amplification conditions are: 1.5 min. 94° C., 2 min. 50° C., 5 min. 60° C. for 35 successive cycles. PCR amplification of the cDNA library resulted in production of a 2 kbp product, which is not observed when either primer is used alone, as shown by agarose gel analysis in FIG. 4 (molecular mass markers are indicated at the left of the figure).
- the PCR amplified fragment is cut with restriction enzymes BamH I and Xba I and following agarose gel purification is ligated into plasmid pUC 118 and transformed into E. coli JM101.
- a large amount of plasmid DNA is prepared for restriction enzyme digestion and sequencing.
- the cDNA lactoferrin insert is cut out with BamH I and Xba I and after agarose gel purification is subjected to digestion with the following restriction enzymes: Bgl I, HgiA I, Pvu II, and Stu I. The digestion is carried out at 37° C. for two hours. Size of the DNA fragments is determined in 2% agarose gel and is shown here as FIG. 8.
- FIG. 8 Size of the DNA fragments is determined in 2% agarose gel and is shown here as FIG. 8.
- FIG. 8 is a restriction fragment map of the cDNA lactoferrin gene.
- the top diagram shows the predicted digestion pattern, while the bottom gel illustrates the size of fragments obtained during digestion.
- Lanes 1 and 7 show molecular mass markers
- lane 3 shows the insert encoding the lactoferrin gene
- lanes 2, 4, 5, and 6 show digestion of the insert with restriction of the enzymes Bgl I, HgiA I, Pvu II and Stu I, respectively.
- the digestion of cDNA lactofernin with restriction enzymes Bgl I, HgiA I and Pvu II generate fragments which follow the pattern of a full length lactoferrin clone.
- Digestion with Stu I generates two (rather than the predicted three) fragments due to methylation of the DNA sequences which overlap the recognition sequence of the restriction endonuclease.
- the cDNA insert encoding the human lactoferrin gene is sequenced in multiple directions using the dideoxy termination method of Sanger, Proc. Nat. Acad. Sci. USA, 74, 5463-5467 (1977), which disclosure is hereby incorporated by reference. Oligomers of 18 bases corresponding to the sequence of 250, 502, 751, 1003, 1252, 1502, and 1751 residues were made and used as primers.
- the nucleotide sequence (Seq. ID No. 1) of the instant clone is shown in FIG. 3.
- the PCR product of Example 10 is digested with BamH I and Xba I, isolated from a 0.7% low melting agarose and ligated into pBlueScriptKS+ (Strata gene Corp.), which is digested with the same restriction enzymes.
- the resulting plasmid is designated pBSlacto.
- the lactoferrin gene containing fragment is then cut out from pBSlacto using Hind III and Sst I and subcloned into pUC118, which is also cut with these enzymes.
- the resulting plasmid is named pUC118-LF and contains the mature lactoferrin gene, that is the nucleotide sequence (Seq. ID No. 1, FIG. 3) encoding the mature lactoferrin protein (Seq. ID No. 2, FIG. 3).
- the signal sequence is added to the 5′-end of the clone using synthetic oligonucleotides based on the signal sequence published by Powell, et al. (1990), supra.
- the oligonucleotides used are shown below:
- Lacto signal I A AGCTT ATGAAACTTGTCTTCCTCGTCCTGTTCTTCCTCGGG Hind III (Seq. ID No. 5)
- Lacto signal II GATCC AGCCAGAGAGAGTCCGAGGGCCCCGAGGAA BamHI (Seq. ID No. 6)
- the Lacto signal I contains a Hind III restriction site and the first 12 codons including the initial methionine.
- Lacto signal II contains a BamH I restriction site and the final 10 codons of the signal peptide sequence.
- the final 9 nucleotides of two oligos are complementary and are annealed, filled-in with the Klenow fragment of DNA polymerase I from E. coli .
- the resulting double-stranded DNA fragment is digested with Hind III and BamH I and ligated into pUC 118-LF, which is similarly digested.
- the resulting plasmid, pUC118-LFS contains a signal sequence with the natural lactoferrin sequence.
- Plasmid pUC118-LFS of Example 12 is digested with Hind III and Xba I and the digested DNA is filled in with Klenow DNA polymerase to produce blunt ends.
- the LF gene with the signal sequence (LFS) is gel purified and ligated to pHIL-DI cut with EcoR I and blunt ended.
- pHILD1-LFS with the correct orientation is identified by screening quick plasmid DNA preps for correct orientation by Stu I digestion. In the correct orientation fragments of size 3.856 Kb and 5.84 Kb are expected. In the wrong orientation fragments of sizes 2.23 Kb and 7.46 Kb are expected.
- Several clones (>12) in the correct orientation are obtained, pooled, and used for Pichia transformation.
- pHILD1-LFS the 5′ untranslated region is as follows: . . . ATTATTCGAAACGAGGAATTAGCTTATG (Seq. ID No. 7).
- Plasmid pHILD1-LFS is divided into two portions. One part is cleaved with Sac I (to direct integration into the AOX1 locus of Pichia strain KM71 [aox1::ARG4, His4]) and the other part is cleaved with Sal I (to direct integration into His4 locus of KM71). Approximately 240 His+ transformants are obtained with Sac I cut DNA, and approximately 120His+ transformants are obtained with Sal I cut DNA.
- Plasmid pPIC9 (Phillips Petroleum Co., Bartlesville, Okla.) is cut with Not I restriction enzyme and alkaline phosphatase treated. Next it is cut with Xho I restriction enzyme. The vector fragment is purified on agarose gel to separate it from the small Aho I-Not I fragment (approx. 43 bp). The purified vector is ligated with alpha mating factor, which is the following 11-mer synthetic oligonucleotide, in which only the top strand is kinased.
- alpha mating factor which is the following 11-mer synthetic oligonucleotide, in which only the top strand is kinased.
- the pPIC9 vector fragment linked with the 11-mer oligonucleotide is gel purified to separate it from excess 11-mer oligonucleotides.
- the resulting gel-purified linked vector fragment which has Xma III and Not I ends, is ligated with the gel-purified Xma III fragment containing the mature lactoferrin gene from Example 13 (Xma III end is compatible with Not I end, thus the Xma III fragment can ligate into the Xma III/Not I ends of the vector).
- the resulting vector is transformed into Pichia pastoris GTS 115 (His4) (Phillips Petroleum Co., Bartlesville, Okla.) by the method of spheroplast.
- the selected transformant cells are used for expression of lactoferrin in a shake flask experiment.
- the selected transformant cells from Example 14 are grown to saturation in 10 ml of buffered minimal glycerol-complex medium in a 50 ml plastic tube in a 30° C. shaker at 300 revolutions/min. for three days.
- the saturated culture is centrifuged for 10 minutes at 3,000 G.
- the cell pellet is resuspended with 2 ml of buffered minimal methanol complex medium and returned to the 30° C. shaker for another three days.
- the supernatant is analyzed for presence of lactoferrin by SDS-PAGE and Western Blot. Lactoferrin is isolated and concentrated from the growth media in one step chromatography.
- Epoxy agarose is saturated with copper-ions (CuSO 4, 5 mg/ml), washed with 50 mM Tris-HCl buffer, pH 8.0. About 200 mg of gel is used for adsorption of lactoferrin from 1 ml of expression medium. The gel is washed with 10 ml of equilibration buffer and lactoferrin is recovered from the gel with 1 ml of 0.2 M ammonium-acetate buffer, pH 3.0.
- proteins are dissolved in 50 ml of SDS sample buffer.
- SDS-PAGE electrophoresis is performed on 9% acrylamide gels according to Laemmli, U.K., (1970) Nature 227, 680-685, the disclosure of which is incorporated by reference herein, and silver stained.
- proteins are transferred to nitrocellulose filters with semi-dry transferring apparatus. The filters are blocked overnight with 5% non-fat dry milk in 50 mM Tris HCl buffer, pH 8.0.
- filters are incubated with rabbit monospecific polyclonal anti-lactoferrin antibodies (1:20,000 dilution) for 2 hr at room temperature, washed four times with 50 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween buffer, pH 8.0, and incubated for 30 min. with protein A conjugated to a horseradish peroxidase. The washes are repeated; the filters are incubated with a chemiluminescent detection kit for 1 min. and exposed to Kodak X-ray film for 1 min.
- FIG. 9 shows the SDS-PAGE analysis (elution from Cu ++ -epoxy agarose) of the thus recovered lactoferrin in lane 4 compared to native lactoferrin from human milk as a positive control in lane 2 and a negative control in lane 3 (medium pass through Cu ++ -epoxy agarose).
- FIG. 10 shows the western blot analysis (elution from Cu ++ -epoxy agarose) of the thus recovered lactoferrin in lane 3 compared to native lactoferrin from human milk as a positive control in lane 1and a negative control in lane 2 (medium pass through Cu ++ -epoxy agarose).
- Molecular mass markers in kDa
Abstract
Reducing the microbial contamination of a composition by treating with lactoferrin.
Description
- This application is a division of application Ser. No. 09/932,190, filed Sep. 17, 2001, which is a division of application Ser. No. 09/421,632, filed Oct. 19, 1999 (now U.S. Pat. No. 6,277,817), which is a division of application Ser. No. 08/724,586, filed Aug. 30, 1996 (now U.S. Pat. No. 6,066,469), which is a continuation of application Ser. No. 08/238,445, filed May 5, 1994 (now abandoned), which is a continuation-in-part of application Ser. No. 08/132,218, filed Oct. 6, 1993 (now Abandoned), which is a continuation of application Ser. No. 07/998,645, filed Dec. 30, 1992 (now abandoned), which is a continuation of application Ser. No. 07/489,186, filed Mar. 8, 1990 (now abandoned), the disclosure of which are incorporated herein by reference.
- The present invention relates generally to the glycoprotein lactoferrin, its production, purification, and uses. BACKGROUND OF THE INVENTION
- Lactoferrin plays an important role in iron transport and utilization in humans. A glycoprotein containing about 6% sugar and having a total molecular weight of about 80,000 daltons, human lactoferrin is capable of binding two ferric ions with high affinity. Although lactoferrin binds iron tightly, the binding is reversible so that the metal is available upon demand to cells with a need for this essential element. The metal is captured by the side chains of specific amino acids: two tyrosines, one histidine and one aspartate which in combination form a cleft in the surface of the protein. That portion of the protein which contains the aforesaid four amino acids and forms the cleft is termed the “iron-binding domain.” Each natural lactoferrin molecule has two iron-binding domains.
- Human milk is high in lactoferrin content. The high degree of iron absorption from human milk is manifested in a low incidence of iron deficiency anemia among breast fed infants compared to infants fed with cow's milk. Accordingly, lactoferrin is a key protein for healthy development of infants. Lactoferrin also plays an important role in cell-mediated host immunity. It is present in high concentrations in all bodily secretions, such as tears, sweat, and ciliated respiratory mucous. Because it sequesters iron, lactoferrin can neutralize pathogenic microorganisms by preventing them from obtaining necessary iron at the site of entry, thereby preventing the spread of infection.
- Although iron is an essential material in humans, excess iron in the body can induce pathological conditions as well. Chronic iron overload, known as hemosiderosis, is characterized by a greater than normal local or generalized deposition of iron within certain body tissues. Lactoferrin helps to manage the balance of free iron in the body to prevent occurrence of such pathological states in healthy individuals.
- The severely limited amount of human milk, the major source of human lactoferrin, restricts lactoferrin production. Furthermore, production of lactoferrin from human milk presents a risk factor of infectious contamination. That is, it could carry with it a potentially lethal contaminant, such as the human immunodeficiency virus (HIV) or another undesirable agent.
- Accordingly, the present invention provides for the cloning and expression of human lactoferrin using recombinant DNA techniques. The lactoferrin of the present invention is free of naturally occurring contaminants, e.g., proteins and viruses, that would be detrimental to the recipient.
- In one embodiment of the present invention there is provided a gene comprising a DNA molecule encoding human lactoferrin protein. More particularly, the DNA molecule comprises the nucleotide sequence (Seq. ID No. 1) and the protein comprises the amino acid sequence (Seq. ID No. 2) as substantially depicted in FIG. 3. An expression system is provided for expressing the gene encoding the protein. Preferably, the expression system is a plasmid. Also described herein, is a host cell line transformed with the gene of the present invention, i.e., the gene encoding human lactoferrin. Preferably the host cell organism is a eukaryotic cell.
- In another embodiment of the invention, there is provided a method of producing human lactoferrin comprising the steps of (a) isolating a gene encoding human lactoferrin; (b) transforming a host cell with the gene; (c) culturing the transformed cells which express the gene product; and (d) collecting lactoferrin from the cells.
- In a further embodiment of the invention there is described a chromatography method for purifying lactoferrin protein, and other proteins, comprising the steps of (a) contacting a substance with a first adsorbent to obtain adsorbed and non-absorbed fractions; (b) eluting the adsorbed fraction with an eluant; and (c) contacting the adsorbed fraction with a second adsorbent, wherein the improvement comprises equilibrating the second adsorbent with the eluant followed by contacting the eluate containing the adsorbed fraction with the second adsorbent.
- In still a further embodiment of the invention there is provided a method for inhibiting microbial growth in a mammal comprising topically or systemically applying to a subject a therapeutically effective amount of lactoferrin having less than about 25% metal loading; a method for inhibiting iron deficiency in a mammal comprising orally administering a therapeutically effective amount of lactoferrin having at least about 35% iron loading; a nutritional supplement comprising an iron-loaded human lactoferrin having at least about 35% metal loading and a nutritionally acceptable carrier or adjuvant; a topical antiseptic comprising a therapeutically effective amount of lactoferrin having less than about 25% metal loading and a pharmaceutically acceptable carrier or diluent; and a method for inhibiting food spoilage comprising adding to the food an effective amount of lactoferrin having less than about 25% metal loading.
- FIG. 1 is a schematic diagram showing the chromatography method of the invention.
- FIG. 2 is a flow chart showing the purification of lactoferrin.
- FIGS. 3a-3 d show the nucleotide sequence (Seq. ID No. 1) and the deduced amino acid sequence (Seq. ID No. 2) of the gene encoding human lactoferrin.
- FIG. 4 is an agarose gel analysis of amplified cDNA coding human lactoferrin.
- FIG. 5 is a map of the pUC118 plasmid.
- FIG. 6 is a map of the pHIL-D1 plasmid.
- FIG. 7 is a map of the pPIC9 plasmid.
- FIG. 8 is a restriction fragment map of the cDNA lactoferrin gene.
- FIG. 9 is an SDS-PAGE analysis of recombinantly expressed lactoferrin according to the present invention.
- FIG. 10 is a Western blot analysis of the recombinantly expressed lactoferrin according to the present invention.
- Lactoferrin is produced in accordance with the present invention using recombinant DNA technology to produce genetically modified DNA that expresses lactoferrin. The recombinant DNA technology described herein is standard technology in the art, such as described by Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989, Chapter 14, which disclosure is hereby incorporated by reference. That is, a polypeptide containing the primary structural conformation of the naturally occurring human lactoferrin protein, having similar biological (i.e. physical) properties is produced. The DNA encoding lactoferrin is preferably from a cDNA library derived from human RNA and ligated to an appropriate expression vector according to standard techniques in the art, such as those disclosed in Davis, et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Co. (1986), which disclosure is hereby incorporated by reference. Preferably, the RNA is isolated from the human mammary gland and the preferred vector is phage λgt11. The cDNA library is screened for positive (DNA carrying the lactoferrin gene) clones using conventional techniques in the art, such as disclosed by Davis, et al., supra, and Rado, et al., Blood, 70:4, 989-993 (October, 1987), which disclosure is hereby incorporated by reference. For example, the cDNA is hybridized to a radiolabeled oligonucleotide probe and the positive clones identified by auto radiography. Preferably, positive clones are identified using lactoferrin antisera, and an appropriate development system, such as an avidin/biotin reaction system. Large numbers of positive clones are then generated by infecting an appropriate bacterial host, such as E. coli Y 1090, using methods that will be readily apparent to the skilled artisan such as described by Davis, et al. (1986), supra. DNA is then isolated from the clones. The cDNA encoding lactoferrin is thereafter cut with an appropriate restriction endonuclease such as EcoRI. The cut DNA encoding lactoferrin is separated by chromatography. Preferably, the separated cDNA is further sub-cloned into another vector, such as the pGEM-4 plasmid, and the inserted cDNA again excised and separated.
- Expression of human lactoferrin according to the present invention is performed using an appropriate host organism, such as the yeastPichia pastoris with an appropriate expression vector such as those driven by the alcohol oxidase promoter and disclosed in Tschopp, Nucleic Acid Research, Vol. 15, pp. 3859-3876, (1987). Other useful hosts include: (i) the yeast Hansenula polymorpha with an appropriate expression vector driven by strong promoters such as methanol oxidase (MOX), dihydroxyacetone synthetase (DAS), or formate dehydrogenase (FMDH) as disclosed in Gellisen, Biotech. Adv., Vol 10, pp. 179-189, (1992) the disclosure of which is incorporated herewith by reference; and (ii) the Spodoptera frugiperda insect cells with an appropriate expression vector such as a Baculovirus expression vector system driven by polyhedrin promoter as disclosed in Gruenwald, Baculovirus Expression Vector System: Procedures & Methods Manual, Second Edition, 1993 by Pharmingen, the disclosure of which is incorporated by reference herein.
- Insertion of the cDNA and expression of the human lactoferrin are carried out using standard recombinant techniques that are readily apparent to one skilled in the art, such as described in Rothstein,Methods in Enzymology, 101, 202-210 (1983); and Tschopp, et al., Bio/Technology, 5, 1305-1308 (1987), which disclosures are hereby incorporated by reference.
- In a preferred embodiment, to obtain the full length cDNA clone of lactoferrin, the gene is amplified from a human mammary gland library using the polymerase chain reaction (PCR) technique. Preferably, amplification of the insert encoding the lactoferrin gene is achieved by using two synthetic oligonucleotide probes corresponding to the amino
acid sequence residues 1 to 9 of the amino terminus andresidues 2070 to 2079 of the carboxy terminus. The amplified insert is cut out with restriction enzymes BamH I and Xba I, then purified, and ligated into an appropriate vector, preferably pBlueScripKS+, which is digested with the same restriction enzymes. The lactoferrin gene is further subcloned into pUC118 vector, cut with Hind III and Sst I to produce a plasmid designated pUC118-LF, containing the coding region for the mature lactoferrin protein. Secretion of the lactoferrin protein may be further enhanced by further manipulations of plasmid pUC118-LF. For example, the following two methods are provided for purposes of illustration. - The first method is the addition of a signal sequence to the 5′ end of the clone. The preferred signal sequence is that described by Powell, et al.,Nucleic Acids Research, 18, 4013 (1990), which disclosure is hereby incorporated by reference. pUC118-LF modified with the aforementioned signal sequence is defined as pUC118-LFS. Preferably, the lactofernin gene, together with the signal sequence is cut from pUC118-LFS and then ligated to pHIL-D1 to produce pHIL-D1-LFS.
- The second, and more preferable, method employs the alpha mating factor pre-pro secretion signal. The mature lactofernin gene is cut from plasmid pUC118-LF and ligated to plasmid pPIC9 which had been previously ligated with an alpha mating factor pre-pro secretion signal.
- An additional embodiment of the present invention relates to fragments of both the lactoferrin gene and lactoferrin. The active sites of the lactoferrin protein are the iron-binding domains. Protein sequences which contain one or more of the iron-binding domains of the lactoferrin protein sequester iron and, therefore, are useful in the antiseptic, dietary supplement, and food-spoilage retardant embodiments of the present invention. The present invention contemplates a (DNA) fragment of the DNA molecule encoding human lactoferrin. The fragment encodes a portion of the human lactoferrin protein containing at least one of the iron-binding domains.
- For example, a DNA fragment of the lactoferrin gene encoding only one iron-binding domain of human lactoferrin can be obtained through a specific design of oligonucleotides for PCR amplification or through an antibody probe screening of a cDNA library, which procedures are readily apparent to one skilled in the art. These fragments are also useful, e.g., as intermediates in the synthesis of the full-length gene and protein.
- Expression of human lactoferrin using the aforementioned modified plasmids is carried out according to techniques that will be readily apparent to the skilled artisan, such as those disclosed in Rothstein (1983) and Tschopp, et al., (1987), supra.
- Purification of the expressed protein according to the present invention is preferably carried out by one of several methods. In one preferred embodiment, a cell-free culture media containing the expressed lactoferrin protein is passed through a filter that retains material having a molecular weight greater than about 10,000 daltons and then sterilizing the retained protein. The material retained by the filter is subjected to a two-step affinity chromatography process. In the first step, the affinity ligand is the reactive dye Cibacron blue F3G-A (color index (C.I.) 61211, λmax 605(374)mn) as disclosed by Bezwoda, et al.,Clin. Chim. Acta., 157, 89-94 (1986); and Chemical Abstracts Service (CAS) No. 12236-82-7, which disclosures are hereby incorporated by reference. Cibacron blue F3G-A can be covalently bound to a cross-linked agarose gel by the triazine coupling method as described by Bohme, et al., J. Chromatography, 69, 209-214 (1972), which disclosure is hereby incorporated by reference. In the second step, controlled-pore glass (CPG) or silicic acid is used to further purify the adsorbed material obtained in the first step.
- In another preferred embodiment, the adsorbed material from the CPG or silicic acid is further chromatographed in a third step using one of the following chromatography techniques before final filtration and sterilization: T-Gel chromatography; immobilized metal-ion affinity chromatography (IMAC) using a metal ion capable of forming a complex with lactoferrin, such as nickel or copper, which can be coupled with an imminodiacetic acid-epoxy activated gel (IDA Me(II)) as described by Sulkowski,Frontiers in Bioprocessing, Sidkar et al., Ed., 343-353 (1990), which disclosure is hereby incorporated by reference; or chromatography with the ligand phenyl glycidyl ether, which can be coupled to a cross-linked agarose gel as described by Janson, et al., Protein Purification Principles High Resolution Methods and Applications, VSH Publishers, New York (1989), which disclosure is hereby incorporated by reference. The two-step and three-step methods previously described are schematically shown by the diagrams in FIG. 2.
- The improved chromatography process of the present invention is useful in purifying proteins, such as lactoferrin produced in accordance with the present invention. As shown in FIG. 1, crude fermentation broth contained in
tank 1 passes topermeable membrane 3, which retains material having a molecular weight greater than 10,000 daltons and passes an ultra filtrate containing water, salts, and low molecular-weight proteins. The retained material is washed with a buffer and further concentrated. The washed material is then applied tochromatography column 5 containing an adsorbent that has been equilibrated with the buffer used to wash the filtered material whilevalve 7 is open and valve 9 is closed. After non-adsorbed material is discharged throughvalve 7,valve 7 is closed and valve 9 opened. Adsorbed material is then eluted, and the eluate passed directly to the second column 11, containing an adsorbent previously equilibrated with the eluant used to elute the adsorbed material. Use of the same medium to elute material from the adsorbent incolumn 5 and equilibrate the adsorbent column 11 avoids the need for timely and involved medium exchange procedures between the two adsorption steps. Passage of the adsorbed material through column 11 occurs while valve 13 is open and valve 15 is closed. Eluting adsorbed material from column 11 occurs while valve 13 is closed and valve 15 is open, thereby passing eluate from column 11 directly to a filter (not shown) capable of retaining material having a molecular weight of at least 10,000 daltons. Although demonstrated for use in purifying lactoferrin, the aforesaid method and apparatus is contemplated in other tandem chromatography procedures that will be readily apparent to the skilled artisan. For example, the invention is useful in purifying proteins with similar hydrophobicity to lactoferrin. - The lactoferrin of the present invention having either no metal loading (iron-free lactoferin, apolactoferrin) or a low metal loading, preferably less than 25%, more preferably less than 20%, most preferably less than 10% of the metal-binding sites occupied, by virtue of being capable of sequestering a significant amount of iron, is useful in applications to individuals where the removal of iron or other transition metals from the individual can have beneficial effects, such as in cosmetics, personal hygiene products, such as feminine douches and mouthwashes, medical and surgical devices and products, topical antiseptics, ophthalmic solutions, oral and intravenous antibiotics, immunopotentiators, antioxidant, and anti-inflammatory and anti-tumor agents.
- Lactoferrin can be used as an antiseptic in accordance with the present invention either alone or in the form of a powder, solution, ointment, aerosol spray, or cream to any part of the subject as an aid in the prevention or treatment of microbial infections. By depriving the surrounding environment of iron, lactoferrin inhibits the growth of microbes, such as bacteria Preferable antiseptics of the present invention include lactoferrin either alone or compounded with carriers such as saline, silica, talcum, stearic acid, its magnesium or calcium salt, polyethyleneglycol, and fatty emulsions and suspensions that will be readily apparent to the skilled artisan. The lactoferrin is preferably present in the antiseptic based on 1 ml of the carrier at 0.1-2 mg, preferably 0.2-2 mg. An effective amount of the lactoferrin varies depending on the individual treated, severity of infection, if any, and the area to which administration is contemplated. Preferably, in treating mammals a twice-daily administration of 0.1-2 mg, more preferably 0.5-2 mg, most preferably 1 mg, of lactofernin per 1 square centimeter effected area is contemplated more preferably 0.1 square centimeter. Lactoferrin can be used as an antiseptic in accordance with the present invention to treat accidental scratches or burns. For example, lactoferrin is applied over the affected area in the form of a 0.1-2 weight %, preferably 0.1-1 weight % solution, or such a solution is used to impregnate a Band-Aid type bandage with lactoferrin. Lactoferrin can be used as an antiseptic in accordance with the present invention to provide prophylaxis in personal hygiene products. For example, the prevention of vaginal infections is accomplished by daily administration of 25-50 mg of lactoferrin in a form of douches or pads. Similarly, the oral infections are prevented by daily administration of 25-50 mg of lactoferrin in a form of mouthwash. Also, lactoferrin provides added protection against sexually transmitted infections when compounded into any device used during sexual activities by either males or females. For example, lactoferrin is added into lubricant to cover condoms at the concentration of 25-50 mg per application. Lactoferrin can be used as antiseptic in accordance with the present invention to impregnate any surgical tools, materials or protective clothing that is used by health care personnel. For example, surgical gloves, masks or linens are covered with a 0.1-2.0% by weight solution of lactoferrin, preferably 0.1-1% by weight. The solution can be applied by a spray, conveniently provided in pressurized aerosol cans or pump bottles.
- Lactoferrin can be useful in the treatment and prevention of opportunistic bacterial, viral, and fungal infections. Opportunistic infections are caused by normally non-pathogenic organisms in patients whose host defense mechanisms have been compromised. By sequestering iron, lactoferrin inhibits the growth of these organisms, making them more susceptible to antibiotic therapy. Depending on the type of infection involved, treatment can involve one or more types of systemic (oral, nasal, intravenous, etc.) or topical administration. Examples of such infections include pneumonia, acquired immune deficiency syndrome (AIDS), candidiasis, diarrhea, and neonatal sepsis. In treating pneumonia, for example pneumonia caused byStreptococcus pneumoniae, antibiotics have minimal impact on mortality during the first five days of illness. By sequestering iron, lactoferrin can inhibit the growth of pneumococcals, and make them more susceptible to antibiotic therapy. Although administration by oral and intravenous routes is contemplated, a simple delivery system of lactoferrin by inhalation involving topical administration to pulmonary membranes is most preferred. Generally, treatment will involve administration three to four times daily of an aqueous solution of lactoferrin in an amount of 100-200 mg per dose for a period of time of 7 to 10 days by inhalation using a known inhaler. A particular cause of opportunistic infections is the lowered host immunity caused by AIDS. Systemic administration of lactoferrin in AIDS patients can help prevent or at least delay the onset of secondary infections. A variety of treatment modalities are contemplated. Intravenous administration of lactoferrin twice daily in an amount of 100-200 mg per injection is recommended for a period of time of one week followed by a one week break. The continuation of this pulse therapy is contemplated for a period of time of three to six months. Depending on the particular infection, an additional localized treatment is also contemplated. For example, in the case of oral candidiasis, the treatment will include administration of lactoferrin as a mouthwash twice daily in an amount of 100-200 mg per dose for a period of time of 7 to 10 days. For a pulmonary infection, such as Pneumocistis carinii, the treatment will involve administration of lactoferrin by inhalation four times daily in an amount of 100-200 mg per dose for a period of time of 7 to 10 days. For Kaposi's sarcoma treatment will involve topical administration of lactoferrin in an ointment, twice daily, in an amount of 50-100 mg per dose for three to four weeks. Fungi infections, depending on the type and location, are treated orally, by intravenous injection, or topical administration. For example, infections, such as vaginal candidiasis, are treated with lactoferrin in a form of douches (vaginal wash) twice daily in an amount of 100-200 mg per dose. Diarrhea, while not usually life threatening, can be dangerous, particularly in infants, because of the potential for fluid imbalance. By virtue of its high affinity for iron, lactoferrin can inhibit the growth of pathogens in the gastrointestinal tract. Treatment of diarrhea will involve oral administration of lactoferin twice daily at an amount of 100-200 mg per dose for a period of time of 7 to 10 days. The treatment of ulcers caused by Helicobacter pylori is also facilitated by the use of lactoferrin. Lactoferrin will not only prevent utilization of iron by the bacteria by sequestering excessive iron from food, it will also deliver the iron to the small intestine where it is recognized by receptors specific to lactoferrin. Treatment will involve oral administration of lactoferrin twice daily in an amount of 200-400 mg per dose for a period of time of 7 to 10 days. Neonatal sepsis, which can coincide with low production levels of lactoferrin in newborn infants, is also subject to treatment by lactoferrin in accordance with the present invention, and particularly in combination with current antibacterial therapy. Treatment will include intravenous administration of lactoferrin twice daily in an amount of 100-200 mg per dose for a period of time of 7 to 10 days. In patients having burns over a large portion of the body, the plasma level of lactoferrin increases 10 to 20 times the normal amount, the body responding to injury by secreting a powerful antimicrobial agent-lactoferrin. Topical administration of lactoferrin to burn patients, by sequestering iron, will prevent development of surface infection. Treatment will involve topical administration of lactofernin in an ointment, cream, or other topical vehicle twice daily in an amount of 50-100 mg per dose for a period of time of three to four weeks. Chronic iron overload, known as hemosiderosis, is characterized by greater than normal iron levels in certain body tissues. When associated with tissue injury, the condition is known as hemochromatosis. Lactoferrin, as the natural chelating agent for iron, offers a viable treatment for such disorders. Preferable treatment involves intravenous or subcutaneous doses of lactoferrin once daily at an amount of 300-500 mg. Lactofernin can also be used to sequester iron implicated in heart disease. By sequestering iron that promotes the oxidation of lipids, which when oxidized can clog arteries, lactoferrin can aid in reducing heart attacks. The prophylactic treatment involves intravenous administration of lactoferrin twice weekly in an amount of 200-500 mg. The treatment is contemplated for high risk patients having high levels of cholesterol. Ischemic heart disease remains the most important cause of morbidity and mortality in the U.S. Over the last decade acute revascularization with thrombolytic drugs has emerged as the standard treatment for patients with acute myocardial infraction. Considerable evidence has emerged over the last decade which indicates that iron may play a key role in pathogenesis of reperfusion injury in the heart. Lactoferrin can sequester large amounts of iron that tend to be released following heart attacks, which thus reduces the amount of iron available for reacting with oxygen to generate free radicals, which cause damage to muscle fibers and cell walls. Preferable treatment involves intravenous administration of lactoferrin immediately after heart attack in an amount of 500-1,000 mg. The treatment of a pneumococcal, streptococcal or staphylococcal infection following trauma of the cornea is also facilitated by lactoferrin. These infections are usually primary causes of corneal ulcers. The treatment will involve topical administration of lactoferrin in an ointment, eye drops or other topical vehicle twice daily in an amount of 10-20 mg per application. Lactoferrin can also be utilized to sequester iron from contact lenses having an application in antiseptic treatment of lenses between wearing times. The effective solution will consist of 0.1-1.0% of lactoferrin in water. The treatment of tumors, such as brain tumors, can also be facilitated by the use of lactoferrin. Newly developed catherization procedures permit the delivery of lactoferrin directly to a blood supply aorta of a tumor, such as a brain tumor, which, by reducing the iron necessary for metabolism of the tumor cells, can prevent the growth of the tumor. Treatment will involve weekly administration of lactoferrin through a microcatheter in an amount of 1-2 grams per dose for a period of time of three to six months. Lactoferrin can also be used as an adjuvant in vaccination. Subunit antigens and peptides made by recombinant DNA technology are not very immunogenic, making their use as vaccines contingent upon the availability of adjuvants that are safe for use in humans and are able to augment sufficiently the immunogenicity of these molecules. Because it modulates a number of immunological responses including myeloid differentiation, modulation of macrophage-mediated cytotoxicity, and regulation of the primary antibody response, lactoferrin can be used as such an adjuvant. The lactoferrin is systemically administered at 100-200 mg per vaccination. Lactoferrin administration before, during, or after vaccination is contemplated based on the specific antigen used for the vaccine.
- The nutritional supplement of the present invention contains an effective amount of lactoferrin loaded with iron, either alone or in combination with one or more nutritionally acceptable carriers or adjuvants. Preferred nutritional supplements include tablets, gelatin capsules, or liquids containing the lactoferrin together with adjuvants or diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine; binders, such as magnesium aluminum silicate, starch, paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone; disintegrants, such as starches, agar, alginic acid or its sodium salt, and effervescent mixtures; as well as absorbents, colorants, flavors and sweeteners. Alternatively, the iron-loaded lactoferrin can be added to foods such as baby formula, cereal, and ice cream to enhance the nutritional value of the food. The preferred amount of iron-loaded lactofernin in the supplement based on the weight of 1 g of the supplement is 5-50 mg, more preferably 20-30 mg, and most preferably 25 mg. An effective daily amount of based on the individual, iron-loaded lactoferrin varies, from about 10-30 mg, preferably 20-30 mg, and more preferably 25 mg. Loading lactoferrin with iron is accomplished by simple titration with, e.g., ferrous ammonium in the presence of bicarbonate, according to methods that will be readily apparent to the skilled artisan. Preferred loading is such that at least about 35%, more preferably at least about 50%, and most preferably at least about 70%, of the metal binding sites are iron bound. Lactoferrin can be applied to food (either solid or liquid) to retard spoilage in accordance with the present invention either alone or compounded with any of the aforesaid nutritionally acceptable carriers or diluents. By sequestering iron, and thereby suppressing its catalytic activity, the lactoferrin reduces the iron available for either microbial multiplication or the production of potentially cell-damaging free-radicals that are formed in iron catalyzed reactions from hydrogen peroxide or superoxide. For example, the lactoferrin is particularly useful in inhibiting rancidity in meat, which is iron-dependent lipid peroxidation. To inhibit microbial growth, particularly in liquid foods such as beer and wine, the lactoferrin can be added directly to the liquid or used to coat filters through which the liquid food passes during processing. An effective amount of the lactoferrin for retarding spoilage varies depending on the type and amount of food contemplated. Preferably, the amount of lactoferrin applied to food in accordance with the present invention varies from 0.1-1 mg/ml of food with which it is mixed, or based on the surface area of the filter or solid food to which it is applied from 0.1-1 mg/cm2. The preferred amount of lactoferrin compounded with a carrier in a food additive for retarding spoilage varies based on 1 ml of the carrier from 0.1-2 mg, preferably 0.2-2 mg.
- The antiseptic, dietary supplement, and food-spoilage retardant of the present invention can be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating, and coating methods.
- The lactoferrin contemplated for use in accordance with the present invention is preferably of human origin, more preferably via DNA recombinant means, but other lactoferrins, such as natural bovine, goat and porcine lactoferrin, isolated and purified using methods applicable to natural human lactoferrin, are contemplated.
- The following non-limiting examples are provided to illustrate the present invention. All parts and percentages are by weight unless indicated otherwise.
- Human lactoferrin is obtained from a genetically altered organism. Using breast tissue excised during the mastectomy of a woman during the eighth month of pregnancy, a human mammary gland genomic library is prepared according to the procedure of Gubler, et al.,Gen., 40, 1-8 (1983), which disclosure is hereby incorporated by reference. A human mammary gland genomic library of cDNA ligated to λgt11 is available from Clontech, Calif. The library is transferred onto agar plates containing a high density of E. coli Y 1090 (Clontech, Calif.) (5×104 plaques per 90 mm plate or 1.4×105 plaques per 150 mm plate). The plates are allowed to stand for 3.5 hours at 42° C. to obtain a lytic growth of the phage. The plates are thereafter overlaid with nitrocellulose filters (Schleiher and Schnell Inc., Wouburn, Mass., under no. BA 85 NC) and heated in an incubator at 37° C. for 3.5 hours.
- Positive clones (i.e., containing the cDNA) are identified on the membranes using rabbit antibody to natural human lactoferrin purified in accordance with Example 9 described hereafter. Nitrocellulose filters are removed from the plates after plaque transferral, and coated with the antibody purified as described under Example 9, which hybridizes with positive plaques. Following removal of excess antibody, positive plaques are developed by first applying an anti-rabbit IgG conjugated with biotin (Sigma Chemical Co., St. Louis, Mo.), and then, following removal of excess biotin conjugate, applying avidin conjugated with horse radish peroxidase (Sigma Chemical Co., St. Louis, Mo.). Finally, the positive plaques are identified in the reaction catalyzed by horse radish peroxidase using as an enzyme substrate 4chloro-1-Naphtol.
- The positive plaques are then used to infectE. coli Y 1090 to produce large amounts of phage in accordance with procedures set forth in Davis, et. al. (1986), supra. The resulting bacteriophage is purified using 10% polyethylene glycol and DNA is isolated from the phage according to the procedures disclosed in Kislow, N. A. R., 14, 6767 (1986), which disclosure is hereby incorporated by reference. Following the procedures in Davis, et al. (1986), supra, the cDNA insert encoding lactoferrin is sub-cloned as follows: the cDNA insert is cut out from the phage DNA using EcoR I and purified using a high resolution ion-exchange chromatography column (GEN-PAK™ Fax, Millipore Corporation, Waters Chromatography Division, Milford, Mass.). The thus purified cDNA insert is ligated using T4 DNA ligase into plasmid pGEM-4 (Promega, Madison, Wis.) as described by Yanish-Perron et al., Gen., 33, 103-109 (1985), which disclosure is hereby incorporated by reference that has been cut using EcoR I using standard techniques. The plasmid containing the insert is then transferred into E. coli JM109 (Promega, Madison, Wis.) as described by Hanahan, J. Mol. Blot, 166, 557 (1983), which disclosure is hereby incorporated by reference. The bacteria are transferred to agar plates containing ampicillin and the positive colonies grown. The plasmid is then isolated and the cDNA insert is cut from the plasmid using EcoR I and purified by ion exchange chromatography as described above.
- The cDNA insert is then ligated into thePichia pastoris expression vector pAO804, so as to be flanked by the 5′ and 3′ regulatory sequences of the methanol-induced alcohol oxidase gene (AOX1) of P. pastoris in accordance with the procedures described by Sreekrishna, et al., Biochemistry, 28, 4117-4125 (1989); and Rothstein, Methods in Enzymology, 101, 202-210 (1983), which disclosures are hereby incorporated by reference. The resulting vector is then transformed into Pichia pastoris GTS115 (His4) by the method of spheroplast as described by Creeg, et al., Mol. Cell. Biol., 5, 3376-3385 (1985), which disclosure is hereby incorporated by reference. The selected transformant cells are grown in 10 ml buffered minimal glycerol-complex medium at 30° C. with shaking for three days. The saturated culture is centrifuged for 10 minutes at 3000 G. The cell pellet is resuspended with 2 ml of buffered minimal methanol-complex medium and returned to the 30° C. shaker for another three days. Cells are pelleted by centrifugation and both the pellet and supernatant are analyzed for the presence of lactoferin. Alternatively, the procedure of Hagenson et al., Enzyme Microb. Technol., 11, 650-656 (1989), which disclosure is hereby incorporated by reference, is followed to grow the cells in a fermentor up to OD600 of about 1.0 then harvested, and washed with and suspended in minimal methanol media at an OD600 about 4.0. The culture is held at 30° C. while maintained at a pH of 5.0 by adding NH3 gas to the air stream. Expressed lactoferrin is recovered from the supernatant of the fermentation media following 15 minutes centrifugation at 5000 rpm using a Beckman J-21B with a Rotor JA 14.
- One liter of the supernatant from Example 1 is adjusted to about 40C. and filtered under pressure through a polysulfone ultrafiltration membrane having a pH operating range of 1-14 on a polypropylene mesh support (PELLICON™ Cassette filter System assembled with Procon pump and PTGC membrane, Millipore Corporation, Milford, Mass.) to retain proteins in excess of about 10,000 molecular weight. Pressure with simultaneous circulation is applied until 900 ml of ultra filtrate is collected. A flow rate of about 100 ml per minute is maintained during the filtration process. The retained material (100 ml) is diluted with 900 ml 20 mM phosphate buffer (pH 7.4) and re-filtered, which is repeated four times (final exchange ratio=10,000). The final material retained is sterilized (0.22 micron GELMAN™ filter).
- Human lactoferrin is purified using affinity chromatography in which the affinity ligand is the reactive dye Cibacron blue F3G-A. The sterilized material obtained in Example 2 is adjusted to a pH of 7.5 and a final concentration of sodium chloride of 0.5 M. This material is then applied onto a column (5 cm×35 cm) packed with cross-linked agarose coupled to the dye (Pharmacia Fine Chemicals, Uppsala, Sweden, BLUE SEPHAROSE™ CL-6B) and previously equilibrated with 50 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) buffer (pH 7.5) containing 0.5 M sodium chloride. Adsorption is performed at a flow rate of 1 ml/min followed by washing the column with 2 bed volumes of the same HEPES buffer. The non-adsorbed fraction is discarded and the adsorbed fraction containing lactoferrin is eluted from the column bed using 2 bed volumes of 50 mM HEPES buffer (pH 7.5) containing 1 M sodium chloride.
- Human lactoferrin is purified using control pore glass (CPG) chromatography. The eluate from Example 3 is applied onto a column (1.2 cm×10 cm) packed with CPG beads (CPG 00350, ElectroNucleonics, Fairfield, N.J.) and previously equilibrated with 50 mM HEPES buffer (pH 7.5) containing 1 M sodium chloride. Adsorption is performed at a flow rate of 1 ml/min followed by washing the column with 2 bed volumes of the same buffer. The non-adsorbed fraction is discarded, and the adsorbed fraction is eluted with 2 bed volumes of 0.25 M tetramethylammonium chloride (TMAC, pH 7.5). The eluate is filtered on a membrane capable of excluding material having a molecular weight greater than 10,000 daltons (AMICON™ YM 10). The filtered material is then sterilized (0.22 micron GELMAN™ filter) and frozen at −200C.
- Human lactoferrin is purified using immobilized metal ion affinity chromatography (IMAC). An imminodiacetic acid-epoxy activated gel (Pharmacia Fine Chemicals, Uppsala, Sweden,
CHELATING SEPHAROSE™ 6B) is washed with water and equilibrated with 0.1 M sodium acetate buffer (pH 4.0) containing 1 M sodium chloride. The gel is then packed into a chromatographic column (1.2 cm×10 cm) and saturated with 4 bed volumes of the same sodium acetate buffer further containing 5 mg/ml of nickel chloride. Excess metal is washed from the column with the sodium acetate buffer, and the gel is equilibrated with 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 2 mM imidazole. - The product of Example 4 is mixed with HEPES, sodium chloride, and imidazole to obtain a pH of 7.0, 20 mM HEPES, 1 M sodium chloride, and 2 mM imidazole. The mixture is applied onto the column at a flow rate of about 1 ml/min followed by,washing the gel with 2 bed volumes of 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 2 mM imidazole. The non-adsorbed fraction is discarded, and the adsorbed fraction containing lactoferrin is eluted using 2 bed volumes of 20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 20 mM imidazole.
- Human lactoferrin is purified using immobilized metal ion affinity chromatography (IMAC). An imminodiacetic acid-epoxy activated gel (Pharmacia Fine Chemicals, Uppsala, Sweden,
CHELATING SEPHAROSE™ 6B) is washed with water and equilibrated with 0.1 M sodium acetate buffer (pH 4.0) containing 1 M sodium chloride. The gel is then packed into a chromatographic column (1.2 cm×10 cm) and saturated with 4 bed volumes of the same sodium acetate buffer further containing 5 mg/ml of copper sulfate. Excess metal is washed from the column with the sodium acetate buffer, and the gel is equilibrated with 50 mM TRIS-HCL buffer (pH 8.0) containing 1 M sodium chloride. - Lactoferrin equilibrated to 50 mM TRIS-HCL pH 8.0, 1 M NaCl is applied onto the column at a flow rate of about 1 ml/min followed by,washing the gel with 2 bed volumes of 50 mM TRIS-HCL buffer (pH 7.0) containing 1 M sodium chloride. The non-adsorbed fraction is discarded, and the adsorbed fraction containing lactoferrin is eluted using 2 bed volumes of 200 mM sodium acetate buffer pH 3.0.
- Human lactoferrin is purified using T-gel affinity chromatography. T-gel adsorbent is prepared according to the procedure described by Porath, et al.,Methods in Enzymology, 44, 19-45 (1976), which disclosure is hereby incorporated by reference, and packed into a column (1.2 cm×10 cm). The final product of Example 4 is adjusted to a pH of 7.5 and a final concentration as follows: 50 mM PIPES buffer (piperazine-N,N′-bis[2-ethanesulfonicacid] and 1,4-piperazinediethanesulfonicacid) buffer and 0.7 M ammonium sulfate. The adjusted material is applied on the column that has been previously equilibrated to 50 mM PIPES buffer (pH 7.5) containing 0.7 M ammonium sulfate with a flow rate of about 1 ml/min. The non-adsorbed fraction containing lactoferrin is adjusted to a concentration of 0.1 M ammonium sulfate and then applied to an identical T-gel column previously equilibrated to 50 mM PIPES buffer (pH 7.5) containing 1.0 M ammonium sulfate. The column is then washed with 7-8 bed volumes of 50 mM PIPES buffer (pH 7.5) containing 1.0 M ammonium sulfate, with lactoferrin being present in the non-adsorbed fraction.
- Human lactoferrin is purified using hydrophobic interaction chromatography on a cross-linked agarose gel coupled to phenyl glycidyl ether (PHENYL SEPHAROSE™ CL-4B, Pharmacia Fine Chemicals, Uppsala, Sweden). The gel is packed into a column and equilibrated to 50 mM PIPES buffer (pH 7.0) containing 1 M ammonium sulfate. The product of Example 4 is adjusted to the equilibrating buffer and applied onto the column at a flow rate of 1 ml/min. The non-adsorbed fraction is discarded and the adsorbed fraction containing lactoferrin is eluted using 2 bed volumes of 50 mM PIPES buffer (pH 7.0).
- Anti-lactoferrin serum is purified by affinity chromatography. The adsorbent substrate for affinity chromatography is prepared by cyanogen bromide activation as described by Axen et al.,Nature, 214, 1302-1304 (1967), which disclosure is hereby incorporated by reference. The substrate (Pharmacia Fine Chemicals, Uppsala, Sweden, CNBR-SEPHAROSE™ 4B) is coupled to human lactoferrin, which acts as the affinity ligand, as follows. One gram of substrate is swelled with 1 mM HCl and washed with the same solvent on a sintered glass filter. Ten mg of natural human lactoferin (Sigma Chemical Co., St. Louis, Mo.) is dissolved in 0.1 M NaHCO3 buffer (pH 8.3) containing 0.5 M sodium chloride (coupling buffer). The resulting solution is mixed with the washed substrate gel for 2 hours, and then mixed with 0.2 M glycine buffer (pH 4.0) for 2 hours. The gel is then washed with coupling buffer, followed by 0.1 M acetate buffer (pH 4.0) containing 0.5 M sodium chloride, followed again by coupling buffer to form the adsorbent. The adsorbent is packed into a column and washed with 20 mM phosphate buffer (pH 7.4) containing 0.5 M sodium chloride. Anti-lactoferrin serum obtained from an inoculated rabbit (Sigma Chemical Co., St. Louis, Mo.) is passed through the column at a flow rate of 1 ml/min and the non-adsorbed material discarded. Adsorbed material containing the purified protein is eluted with 2 bed volumes of 0.2 M glycine buffer (pH 2.0) containing 0.5 M of sodium chloride. The eluate is neutralized with 0.1N NaOH to obtain pH 7.5 and then sterilized (0.22 micron GELMAN™ filter) and frozen at −20° C.
- The human lactoferrin gene is cloned by PCR amplification from a human mammary gland library in λgt11. Phage DNA is prepared from the library by using standard procedures described in Maniatis (1989), supra. Oligonucleotide primers are prepared to the 5′- and 3′-ends of the gene based on the sequence published by Powell, et al. (1990), supra. The 5′-oligo is designed to begin at the coding sequence for the first glycine of the mature lactoferrin protein as defined by Powell, et al. (1990), supra, and the 3′-oligo included the lactoferrin stop codon and 21 nucleotides beyond. These oligonucleotides are prepared on an Applied Biosystems DNA synthesizer Model 380A and were obtained from Roswell Park Cancer Institute, Buffalo, N.Y. Each oligo also created a restriction endonuclease recognition site, the 5′-oligo contained a BamH I site and the 3′-oligo an Xba I site. The sequences of these oligonucleotides are given below:
- 5′-oligo AGCGGATCCGGCCGTAGGAGAAGGAGTGTTCAGTGG (Seq. ID No.3) BamH I
- 3′-oligo CGATCTAGATTACTTCCTGAGGAATCCACAGGC (Seq. ID No.4) Xba I
- PCR amplification of the cDNA library using these oligonucleotide primers is performed with a temperature cycler (Genetic Thermal Cycler, Precision Scientific), utilizing Tag polymerase. Amplification conditions are: 1.5 min. 94° C., 2 min. 50° C., 5 min. 60° C. for 35 successive cycles. PCR amplification of the cDNA library resulted in production of a 2 kbp product, which is not observed when either primer is used alone, as shown by agarose gel analysis in FIG. 4 (molecular mass markers are indicated at the left of the figure).
- The PCR amplified fragment is cut with restriction enzymes BamH I and Xba I and following agarose gel purification is ligated into plasmid pUC 118 and transformed intoE. coli JM101. A large amount of plasmid DNA is prepared for restriction enzyme digestion and sequencing. The cDNA lactoferrin insert is cut out with BamH I and Xba I and after agarose gel purification is subjected to digestion with the following restriction enzymes: Bgl I, HgiA I, Pvu II, and Stu I. The digestion is carried out at 37° C. for two hours. Size of the DNA fragments is determined in 2% agarose gel and is shown here as FIG. 8. FIG. 8 is a restriction fragment map of the cDNA lactoferrin gene. The top diagram shows the predicted digestion pattern, while the bottom gel illustrates the size of fragments obtained during digestion.
Lanes lane 3 shows the insert encoding the lactoferrin gene, andlanes - The cDNA insert encoding the human lactoferrin gene is sequenced in multiple directions using the dideoxy termination method of Sanger,Proc. Nat. Acad. Sci. USA, 74, 5463-5467 (1977), which disclosure is hereby incorporated by reference. Oligomers of 18 bases corresponding to the sequence of 250, 502, 751, 1003, 1252, 1502, and 1751 residues were made and used as primers. The nucleotide sequence (Seq. ID No. 1) of the instant clone is shown in FIG. 3.
- The PCR product of Example 10 is digested with BamH I and Xba I, isolated from a 0.7% low melting agarose and ligated into pBlueScriptKS+ (Strata gene Corp.), which is digested with the same restriction enzymes. The resulting plasmid is designated pBSlacto. The lactoferrin gene containing fragment is then cut out from pBSlacto using Hind III and Sst I and subcloned into pUC118, which is also cut with these enzymes. The resulting plasmid is named pUC118-LF and contains the mature lactoferrin gene, that is the nucleotide sequence (Seq. ID No. 1, FIG. 3) encoding the mature lactoferrin protein (Seq. ID No. 2, FIG. 3).
- To allow secretion of the lactoferrin protein, the signal sequence is added to the 5′-end of the clone using synthetic oligonucleotides based on the signal sequence published by Powell, et al. (1990), supra. The oligonucleotides used are shown below:
- Lacto signal I: AAGCTTATGAAACTTGTCTTCCTCGTCCTGTTCTTCCTCGGG Hind III (Seq. ID No. 5)
- Lacto signal II:GATCCAGCCAGAGAGAGTCCGAGGGCCCCGAGGAA BamHI (Seq. ID No. 6)
- The Lacto signal I contains a Hind III restriction site and the first 12 codons including the initial methionine. Lacto signal II contains a BamH I restriction site and the final 10 codons of the signal peptide sequence. The final 9 nucleotides of two oligos are complementary and are annealed, filled-in with the Klenow fragment of DNA polymerase I fromE. coli. The resulting double-stranded DNA fragment is digested with Hind III and BamH I and ligated into pUC 118-LF, which is similarly digested. The resulting plasmid, pUC118-LFS, contains a signal sequence with the natural lactoferrin sequence.
- Plasmid pUC118-LFS of Example 12 is digested with Hind III and Xba I and the digested DNA is filled in with Klenow DNA polymerase to produce blunt ends. The LF gene with the signal sequence (LFS) is gel purified and ligated to pHIL-DI cut with EcoR I and blunt ended. pHILD1-LFS with the correct orientation is identified by screening quick plasmid DNA preps for correct orientation by Stu I digestion. In the correct orientation fragments of size 3.856 Kb and 5.84 Kb are expected. In the wrong orientation fragments of sizes 2.23 Kb and 7.46 Kb are expected. Several clones (>12) in the correct orientation are obtained, pooled, and used for Pichia transformation. In pHILD1-LFS the 5′ untranslated region is as follows: . . . ATTATTCGAAACGAGGAATTAGCTTATG (Seq. ID No. 7). The nucleotide composition of −1 to −25 position is AT:GC=68:32, which is in the preferred range for Pichia expression.
- Plasmid pHILD1-LFS is divided into two portions. One part is cleaved with Sac I (to direct integration into the AOX1 locus of Pichia strain KM71 [aox1::ARG4, His4]) and the other part is cleaved with Sal I (to direct integration into His4 locus of KM71). Approximately 240 His+ transformants are obtained with Sac I cut DNA, and approximately 120His+ transformants are obtained with Sal I cut DNA.
- Plasmid pPIC9 (Phillips Petroleum Co., Bartlesville, Okla.) is cut with Not I restriction enzyme and alkaline phosphatase treated. Next it is cut with Xho I restriction enzyme. The vector fragment is purified on agarose gel to separate it from the small Aho I-Not I fragment (approx. 43 bp). The purified vector is ligated with alpha mating factor, which is the following 11-mer synthetic oligonucleotide, in which only the top strand is kinased.
- (P-TCGAGAAAAGA CTTTTCTCCGG-OH) (Seq. ID No. 8)
- The pPIC9 vector fragment linked with the 11-mer oligonucleotide is gel purified to separate it from excess 11-mer oligonucleotides. The resulting gel-purified linked vector fragment, which has Xma III and Not I ends, is ligated with the gel-purified Xma III fragment containing the mature lactoferrin gene from Example 13 (Xma III end is compatible with Not I end, thus the Xma III fragment can ligate into the Xma III/Not I ends of the vector). The resulting vector is transformed intoPichia pastoris GTS 115 (His4) (Phillips Petroleum Co., Bartlesville, Okla.) by the method of spheroplast. The selected transformant cells are used for expression of lactoferrin in a shake flask experiment.
- The selected transformant cells from Example 14 are grown to saturation in 10 ml of buffered minimal glycerol-complex medium in a 50 ml plastic tube in a 30° C. shaker at 300 revolutions/min. for three days. The saturated culture is centrifuged for 10 minutes at 3,000 G. The cell pellet is resuspended with 2 ml of buffered minimal methanol complex medium and returned to the 30° C. shaker for another three days. The supernatant is analyzed for presence of lactoferrin by SDS-PAGE and Western Blot. Lactoferrin is isolated and concentrated from the growth media in one step chromatography. Epoxy agarose is saturated with copper-ions (
CuSO 4, 5 mg/ml), washed with 50 mM Tris-HCl buffer, pH 8.0. About 200 mg of gel is used for adsorption of lactoferrin from 1 ml of expression medium. The gel is washed with 10 ml of equilibration buffer and lactoferrin is recovered from the gel with 1 ml of 0.2 M ammonium-acetate buffer, pH 3.0. - After lyophilization, proteins are dissolved in 50 ml of SDS sample buffer. SDS-PAGE electrophoresis is performed on 9% acrylamide gels according to Laemmli, U.K., (1970) Nature 227, 680-685, the disclosure of which is incorporated by reference herein, and silver stained. After SDS-PAGE electrophoresis, proteins are transferred to nitrocellulose filters with semi-dry transferring apparatus. The filters are blocked overnight with 5% non-fat dry milk in 50 mM Tris HCl buffer, pH 8.0. After a brief washing with 50 mM Tris-HCl buffer, pH 8.0, filters are incubated with rabbit monospecific polyclonal anti-lactoferrin antibodies (1:20,000 dilution) for 2 hr at room temperature, washed four times with 50 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween buffer, pH 8.0, and incubated for 30 min. with protein A conjugated to a horseradish peroxidase. The washes are repeated; the filters are incubated with a chemiluminescent detection kit for 1 min. and exposed to Kodak X-ray film for 1 min.
- FIG. 9 shows the SDS-PAGE analysis (elution from Cu++-epoxy agarose) of the thus recovered lactoferrin in
lane 4 compared to native lactoferrin from human milk as a positive control inlane 2 and a negative control in lane 3 (medium pass through Cu++-epoxy agarose). FIG. 10 shows the western blot analysis (elution from Cu++-epoxy agarose) of the thus recovered lactoferrin inlane 3 compared to native lactoferrin from human milk as a positive control in lane 1and a negative control in lane 2 (medium pass through Cu++-epoxy agarose). Molecular mass markers (in kDa) are indicated at the left of FIGS. 9 and 10. The results show that the expressed -
1 8 2086 base pairs nucleic acid single linear DNA (genomic) NO NO CDS 1..2086 1 GGA TCC GGC CGT AGG AGA AGG AGT GTT CAG TGG TGC GCC GTA TCC CAA 48 Gly Ser Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln 1 5 10 15 CCC GAG GCC ACA AAA TGC TTC CAA TGG CAA AGG AAT ATG AGA AAA GTG 96 Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val 20 25 30 CGT GGC CCT CCT GTC AGC TGC ATA AAG AGA GAC TCC CCC ATC CAG TGT 144 Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys 35 40 45 ATC CAG GCC ATT GCG GAA AAC AGG GCC GAT GCT GTG ACC CTT GAT GGT 192 Ile Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly 50 55 60 GGT TTC ATA TAC GAG GCA GGC CTG GCC CCC TAC AAA CTG CGA CCT GTA 240 Gly Phe Ile Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val 65 70 75 80 GCG GCG GAA GTC TAC GGG ACC GAA AGA CAG CCA CGA ACT CAC TAT TAT 288 Ala Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr 85 90 95 GCC GTG GCT GTG GTG AAG AAG GGC GGC AGC TTT CAG CTG AAC GAA CTG 336 Ala Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu 100 105 110 CAA GGT CTG AAG TCC TGC CAC ACA GGC CTT CGC AGG ACC GCT GGA TGG 384 Gln Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly Trp 115 120 125 AAT GTC CCT ATA GGG ACA CTT CGT CCA TTC TTG AAT TGG ACG GGT CCA 432 Asn Val Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly Pro 130 135 140 CCT GAG CCC ATT GAG GCA GCT GTG GCC AGG TTC TTC TCA GCC AGC TGT 480 Pro Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser Cys 145 150 155 160 GTT CCC GGT GCA GAT AAA GGA CAG TTC CCC AAC CTG TGT CGC CTG TGT 528 Val Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg Leu Cys 165 170 175 GCG GGG ACA GGG GAA AAC AAA TGT GCC TTC TCC TCC CAG GAA CCG TAC 576 Ala Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr 180 185 190 TTC AGC TAC TCT GGT GCC TTC AAG TGT CTG AGA GAC GGG GCT GGA GAC 624 Phe Ser Tyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp 195 200 205 GTG GCT TTT ATC AGA GAG AGC ACA GTG TTT GAG GAC CTG TCA GAC GAG 672 Val Ala Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu 210 215 220 GCT GAA AGG GAC GAG TAT GAG TTA CTC TGC CCA GAC AAC ACT CGG AAG 720 Ala Glu Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys 225 230 235 240 CCA GTG GAC AAG TTC AAA GAC TGC CAT CTG GCC CGG GTC CCT TCT CAT 768 Pro Val Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser His 245 250 255 GCC GTT GTG GCA CGA AGT GTG AAT GGC AAG GAG GAT GCC ATC TGG AAT 816 Ala Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp Asn 260 265 270 CTT CTC CGC CAG GCA CAG GAA AAG TTT GGA AAG GAC AAG TCA CCG AAA 864 Leu Leu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro Lys 275 280 285 TTC CAG CTC TTT GGC TCC CCT AGT GGG CAG AAA GAT CTG CTG TTC AAG 912 Phe Gln Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu Leu Phe Lys 290 295 300 GAC TCT GCC ATT GGG TTT TCG AGG GTG CCC CCG AGG ATA GAT TCT GGG 960 Asp Ser Ala Ile Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly 305 310 315 320 CTG TAC CTT GGC TCC GGC TAC TTC ACT GCC ATC CAG AAC TTG AGG AAA 1008 Leu Tyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys 325 330 335 AGT GAG GAG GAA GTG GCT GCC CGG CGT GCG CGG GTC GTG TGG TGT GCG 1056 Ser Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala 340 345 350 GTG GGC GAG CAG GAG CTG CGC AAG TGT AAC CAG TGG AGT GGC TTG AGC 1104 Val Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu Ser 355 360 365 GAA GGC AGC GTG ACC TGC TCC TCG GCC TCC ACC ACA GAG GAC TGC ATC 1152 Glu Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys Ile 370 375 380 GCC CTG GTG CTG AAA GGA GAA GCT GAT GCC ATG AGT TTG GAT GGA GGA 1200 Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly 385 390 395 400 TAT GTG TAC ACT GCA GGC AAA TGT GGT TTG GTG CCT GTC CTG GCA GAG 1248 Tyr Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu 405 410 415 AAC TAC AAA TCC CAA CAA AGC AGT GAC CCT GAT CCT AAC TGT GTG GAT 1296 Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp 420 425 430 AGA CCT GTG GAA GGA TAT CTT GCT GTG GCG GTG GTT AGG AGA TCA GAC 1344 Arg Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp 435 440 445 ACT AGC CTT ACC TGG AAC TCT GTG AAA GGC AAG AAG TCC TGC CAC ACC 1392 Thr Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr 450 455 460 GCC GTG GAC AGG ACT GCA GGC TGG AAT ATC CCC ATG GGC CTG CTC TTC 1440 Ala Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe 465 470 475 480 AAC CAG ACG GGC TCC TGC AAA TTT GAT GAA TAT TTC AGT CAA AGC TGT 1488 Asn Gln Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gln Ser Cys 485 490 495 GCC CCT GGG TCT GAC CCG AGA TCT AAT CTC TGT GCT CTG TGT ATT GGC 1536 Ala Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile Gly 500 505 510 GAC GAG CAG GGT GAG AAT AAG TGC GTG CCC AAC AGC AAC GAG AGA TAC 1584 Asp Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg Tyr 515 520 525 TAC GGC TAC ACT GGG GCT TTC CGG TGC CTG GCT GAG AAT GCT GGA GAC 1632 Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly Asp 530 535 540 GTT GCA TTT GTG AAA GAT GTC ACT GTC TTG CAG AAC ACT GAT GGA AAT 1680 Val Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn 545 550 555 560 AAC AAT GAG GCA TGG GCT AAG GAT TTG AAG CTG GCA GAC TTT GCG CTG 1728 Asn Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu 565 570 575 CTG TGC CTC GAT GGC AAA CGG AAG CCT GTG ACT GAG GCT AGA AGC TGC 1776 Leu Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys 580 585 590 CAT CTT GCC ATG GCC CCG AAT CAT GCC GTG GTG TCT CGG ATG GAT AAG 1824 His Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp Lys 595 600 605 GTG GAA CGC CTG AAA CAG GTG TTG CTC CAC CAA CAG GCT AAA TTT GGG 1872 Val Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe Gly 610 615 620 AGA AAT GGA TCT GAC TGC CCG GAC AAG TTT TGC TTA TTC CAG TCT GAA 1920 Arg Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser Glu 625 630 635 640 ACC AAA AAC CTT CTG TTC AAT GAC AAC ACT GAG TGT CTG GCC AGA CTC 1968 Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg Leu 645 650 655 CAT GGC AAA ACA ACA TAT GAA AAA TAT TTG GGA CCA CAG TAT GTC GCA 2016 His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala 660 665 670 GGC ATT ACT AAT CTG AAA AAG TGC TCA ACC TCC CCC CTC CTG GAA GCC 2064 Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala 675 680 685 TGT GAA TTC CTC AGG AAG TAA A 2086 Cys Glu Phe Leu Arg Lys * 690 695 694 amino acids amino acid linear protein 2 Gly Ser Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln 1 5 10 15 Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val 20 25 30 Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys 35 40 45 Ile Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly 50 55 60 Gly Phe Ile Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val 65 70 75 80 Ala Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr 85 90 95 Ala Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu 100 105 110 Gln Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly Trp 115 120 125 Asn Val Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly Pro 130 135 140 Pro Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser Cys 145 150 155 160 Val Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg Leu Cys 165 170 175 Ala Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr 180 185 190 Phe Ser Tyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp 195 200 205 Val Ala Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu 210 215 220 Ala Glu Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys 225 230 235 240 Pro Val Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser His 245 250 255 Ala Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp Asn 260 265 270 Leu Leu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro Lys 275 280 285 Phe Gln Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu Leu Phe Lys 290 295 300 Asp Ser Ala Ile Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly 305 310 315 320 Leu Tyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys 325 330 335 Ser Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala 340 345 350 Val Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu Ser 355 360 365 Glu Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys Ile 370 375 380 Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly 385 390 395 400 Tyr Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu 405 410 415 Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp 420 425 430 Arg Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp 435 440 445 Thr Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr 450 455 460 Ala Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe 465 470 475 480 Asn Gln Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gln Ser Cys 485 490 495 Ala Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile Gly 500 505 510 Asp Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg Tyr 515 520 525 Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly Asp 530 535 540 Val Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn 545 550 555 560 Asn Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu 565 570 575 Leu Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys 580 585 590 His Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp Lys 595 600 605 Val Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe Gly 610 615 620 Arg Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser Glu 625 630 635 640 Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg Leu 645 650 655 His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala 660 665 670 Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala 675 680 685 Cys Glu Phe Leu Arg Lys 690 695 36 base pairs nucleic acid single linear protein NO NO 3 AGCGGATCCG GCCGTAGGAG AAGGAGTGTT CAGTGG 36 33 base pairs nucleic acid single linear DNA (genomic) NO NO 4 CGATCTAGAT TACTTCCTGA GGAATCCACA GGC 33 42 base pairs nucleic acid single linear DNA (genomic) NO NO 5 AAGCTTATGA AACTTGTCTT CCTCGTCCTG TTCTTCCTCG GG 42 35 base pairs nucleic acid single linear protein NO NO 6 GATCCAGCCA GAGAGAGTCC GAGGGCCCCG AGGAA 35 28 base pairs nucleic acid single linear DNA (genomic) NO NO 7 ATTATTCGAA ACGAGGAATT AGCTTATG 28 22 base pairs nucleic acid single linear DNA (genomic) NO NO 8 TCGAGAAAAG ACTTTTCTCC GG 22
Claims (12)
1. A method for reducing the microbial contamination of a meat product, comprising treating the meat product with a sufficient amount of lactoferrin to reduce microbial contamination.
2. A method for reducing the microbial contamination of a meat product, comprising treating the meat product with a sufficient amount of isolated lactoferrin to reduce microbial contamination.
3. A method for reducing the microbial contamination of a meat product, comprising treating the meat product with a sufficient amount of lactoferrin mixed with a carrier to reduce microbial contamination.
4. A method for reducing the microbial contamination of a meat product, comprising treating the meat product with a sufficient amount of lactofernin mixed with a naturally occuring carrier to reduce microbial contamination.
5. A method for reducing the microbial contamination of a meat product, comprising treating the meat product with a sufficient amount of isolated lactoferrin mixed with a naturally occuring carrier to reduce microbial contamination.
6. A method in accordance with claim 1 , wherein said lactoferrin is a recombinant lactoferrin.
7. A method in accordance with claim 3 , wherein said lactoferrin is a recombinant lactofernin.
8. A method for reducing the microbial contamination of a composition subject to microbial contamination comprising treating the composition with a sufficient amount of lactoferrin to reduce microbial contamination.
9. A method for reducing the microbial contamination of a composition subject to microbial contamination comprising treating the composition with a sufficient amount of isolated lactoferrin to reduce microbial contamination.
10. A method for reducing the microbial contamination of a composition subject to microbial contamination comprising treating the composition with a sufficient amount of isolated lactoferrin compounded with a carrier to reduce microbial contamination.
11. A method for reducing the microbial contamination of a composition subject to microbial contamination comprising treating the composition with a sufficient amount of isolated lactoferrin bound with a naturally occuring carrier to reduce microbial contamination.
12. A method for retarding food-spoilage comprising applying to food an effective amount of lactoferrin having less than 25% metal loading.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/023,096 US20020160941A1 (en) | 1990-03-08 | 2001-12-18 | Treating compositions with lactoferrin |
US11/810,477 US20070259007A1 (en) | 1999-10-19 | 2007-06-06 | Lactoferrin: an adjuvant for vaccines |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US48918690A | 1990-03-08 | 1990-03-08 | |
US99864592A | 1992-12-30 | 1992-12-30 | |
US23844594A | 1994-05-05 | 1994-05-05 | |
US08/724,586 US6066469A (en) | 1990-03-08 | 1996-09-30 | Cloning, expression, and uses of human lactoferrin |
US09/421,632 US6277817B1 (en) | 1990-03-08 | 1999-10-19 | Human lactoferrin |
US09/932,190 US6455687B1 (en) | 1990-03-08 | 2001-08-17 | Human lactoferrin |
US10/023,096 US20020160941A1 (en) | 1990-03-08 | 2001-12-18 | Treating compositions with lactoferrin |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/932,190 Division US6455687B1 (en) | 1990-03-08 | 2001-08-17 | Human lactoferrin |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/810,477 Continuation-In-Part US20070259007A1 (en) | 1999-10-19 | 2007-06-06 | Lactoferrin: an adjuvant for vaccines |
Publications (1)
Publication Number | Publication Date |
---|---|
US20020160941A1 true US20020160941A1 (en) | 2002-10-31 |
Family
ID=27494955
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/724,586 Expired - Fee Related US6066469A (en) | 1990-03-08 | 1996-09-30 | Cloning, expression, and uses of human lactoferrin |
US09/421,632 Expired - Fee Related US6277817B1 (en) | 1990-03-08 | 1999-10-19 | Human lactoferrin |
US09/932,190 Expired - Fee Related US6455687B1 (en) | 1990-03-08 | 2001-08-17 | Human lactoferrin |
US10/023,096 Abandoned US20020160941A1 (en) | 1990-03-08 | 2001-12-18 | Treating compositions with lactoferrin |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/724,586 Expired - Fee Related US6066469A (en) | 1990-03-08 | 1996-09-30 | Cloning, expression, and uses of human lactoferrin |
US09/421,632 Expired - Fee Related US6277817B1 (en) | 1990-03-08 | 1999-10-19 | Human lactoferrin |
US09/932,190 Expired - Fee Related US6455687B1 (en) | 1990-03-08 | 2001-08-17 | Human lactoferrin |
Country Status (1)
Country | Link |
---|---|
US (4) | US6066469A (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030229011A1 (en) * | 2001-12-28 | 2003-12-11 | Braun Steven O. | Humanized lactoferrin and uses thereof |
WO2005079582A1 (en) * | 2004-02-24 | 2005-09-01 | Campina B.V. | Antimicrobial lactoferrin compositions for surfaces, cavities, and foodstuff |
EP1899375A1 (en) * | 2005-05-13 | 2008-03-19 | Array Biopharma Inc. | New purification method of lactoferrin |
US20080318834A1 (en) * | 2004-02-24 | 2008-12-25 | Campina B.V. | Antimicrobial Lactoferrin Compositions for Surfaces, Cavities, and Foodstuff |
WO2009102677A1 (en) * | 2008-02-11 | 2009-08-20 | Tabatchnick Fine Foods, Inc. | A nutritionally enhanced therapeutic preventative food supplement and method of making same |
US20090291122A1 (en) * | 2006-06-01 | 2009-11-26 | Sano Medical Bvba | Wound Care Treatment Product |
US20100198946A1 (en) * | 2009-02-05 | 2010-08-05 | At&T Mobility Ii Llc | SYSTEM AND METHOD FOR QUALITY OF SERVICE (QoS) PARAMETER CHANGE |
US20130323314A1 (en) * | 2011-12-30 | 2013-12-05 | University Of Hyderabad | Novel Nanoparticles of Lactoferrin Useful for Preparing a Pharmaceutical Composition Facilitating Easy Delivery of the Drug and a Process for Preparing the Same |
US9149035B2 (en) | 2005-12-14 | 2015-10-06 | Convatec Technologies, Inc. | Antimicrobial composition |
US11135315B2 (en) | 2010-11-30 | 2021-10-05 | Convatec Technologies Inc. | Composition for detecting biofilms on viable tissues |
US11286601B2 (en) | 2012-12-20 | 2022-03-29 | Convatec Technologies, Inc. | Processing of chemically modified cellulosic fibres |
Families Citing this family (45)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL94183A (en) * | 1989-05-05 | 2003-09-17 | Baylor College Medicine | cDNA SEQUENCE CODING FOR HUMAN LACTOFERRIN PROTEIN OR PORTION THEREOF AND LACTOFERRIN PROTEIN PRODUCED FROM SAID SEQUENCE |
AU6307698A (en) * | 1997-02-03 | 1998-08-25 | Pharming Bv | Useful properties of human lactoferrin and variants thereof |
DK0988861T3 (en) * | 1998-08-17 | 2004-04-19 | Pfizer Prod Inc | Stabilized protein preparations |
US6399570B1 (en) * | 1999-02-05 | 2002-06-04 | Agennix, Inc. | Antimicrobial/endotoxin neutralizing polypeptide |
US6172040B1 (en) * | 1999-05-28 | 2001-01-09 | A. Satyanarayan Naidu | Immobilized lactoferrin antimicrobial agents and the use thereof |
EP1068871A1 (en) * | 1999-07-07 | 2001-01-17 | Jean-Paul Perraudin | Novel methods and medicament for treating infections diseases involving microbial biofilms |
AU7042000A (en) * | 1999-10-19 | 2001-05-14 | Easy Bio System, Inc. | Recombinant yeast strain for expressing human lactoferrin, n-lobe lactoferrin, c-lobe lactoferrin and process for preparation recombinant human lactoferrin, n-lobe lactoferrin, c-lobe lactoferrin using the same |
WO2006063329A2 (en) * | 2004-12-10 | 2006-06-15 | Board Of Regents Of The University Of Texas System | Enhanced protection against mycobacterium tuberculosis |
US20080050503A1 (en) * | 2000-05-02 | 2008-02-28 | Ning Huang | Expression of human milk proteins in transgenic plants |
US7417178B2 (en) * | 2000-05-02 | 2008-08-26 | Ventria Bioscience | Expression of human milk proteins in transgenic plants |
US7138150B2 (en) * | 2000-05-02 | 2006-11-21 | Ventria Bioscience | Method of making an anti-infective composition for treating oral infections |
US20070031440A1 (en) * | 2001-08-30 | 2007-02-08 | Prior Christopher P | Modified transferin-antibody fusion proteins |
US8129504B2 (en) | 2001-08-30 | 2012-03-06 | Biorexis Technology, Inc. | Oral delivery of modified transferrin fusion proteins |
US7176278B2 (en) * | 2001-08-30 | 2007-02-13 | Biorexis Technology, Inc. | Modified transferrin fusion proteins |
WO2003020746A1 (en) * | 2001-08-30 | 2003-03-13 | Biorexis Pharmaceutical Corporation | Modified transferrin fusion proteins |
US20030226155A1 (en) * | 2001-08-30 | 2003-12-04 | Biorexis Pharmaceutical Corporation | Modified transferrin-antibody fusion proteins |
WO2003049455A1 (en) * | 2001-11-30 | 2003-06-12 | Zaxel Systems, Inc. | Image-based rendering for 3d object viewing |
EP1545595B1 (en) * | 2002-08-30 | 2010-07-07 | Biorexis Pharmaceutical Corporation | Modified transferrin fusion proteins comprising duplicate transferrin amino or carboxy terminal domains |
US20060105387A1 (en) * | 2002-08-30 | 2006-05-18 | Prior Christopher P | Transferrin fusion proteins libraries |
DK1545587T3 (en) * | 2002-09-16 | 2011-05-09 | Agennix Inc | Lactoferrin compositions and methods for the treatment of diabetic wounds |
CA2508912A1 (en) * | 2002-12-06 | 2004-06-24 | Agennix Incorporated | Oral lactoferrin in the treatment of sepsis |
AU2003296447A1 (en) * | 2002-12-10 | 2004-06-30 | Agennix Incorporated | Lactoferrin as an agent in the prevention of organ transplant rejection and graft-versus-host-disease |
AU2003293500A1 (en) * | 2002-12-12 | 2004-07-09 | Agennix Incorporated | Lactoferrin in the reduction of pain |
US20070060512A1 (en) * | 2003-03-04 | 2007-03-15 | Homayoun Sadeghi | Dipeptidyl-peptidase protected protein |
KR100468596B1 (en) * | 2003-04-04 | 2005-01-27 | (주) 디지탈바이오텍 | Method for production of human lactoferrin using methylotrophic yeast, Pichia pastoris, human lactoferrin manufactured thereby and Pichia pastoris |
US7379700B2 (en) * | 2003-05-06 | 2008-05-27 | Canon Kabushiki Kaisha | Image reading apparatus with rotatable internal and external guides |
WO2004103285A2 (en) * | 2003-05-14 | 2004-12-02 | Agennix Incorporated | Lactoferrin in the treatment of diabetes mellitus |
US8105615B2 (en) * | 2003-06-06 | 2012-01-31 | Agennix Incorporated | Lactoferrin as an adjuvant in cancer vaccines |
US20040259770A1 (en) * | 2003-06-19 | 2004-12-23 | Rush University Medical Center | Method for determining leukocyte activation |
US20060205037A1 (en) * | 2003-08-28 | 2006-09-14 | Homayoun Sadeghi | Modified transferrin fusion proteins |
WO2005021579A2 (en) * | 2003-08-28 | 2005-03-10 | Biorexis Pharmaceutical Corporation | Epo mimetic peptides and fusion proteins |
US7125963B2 (en) * | 2004-03-03 | 2006-10-24 | En N Tech Inc | Treatments for contaminant reduction in lactoferrin preparations and lactoferrin containing compositions |
WO2006047744A2 (en) * | 2004-10-26 | 2006-05-04 | Agennix Incorporated | Compositions of lactoferrin related peptides and uses thereof |
US7956031B2 (en) * | 2005-05-31 | 2011-06-07 | Naidu Lp | Metallo-lactoferrin-coenzyme compositions for trigger and release of bioenergy |
ITMI20052351A1 (en) * | 2005-12-09 | 2007-06-10 | Microbo Srl | NEW PHARMACEUTICAL USE OF TRANSFERRIN AND DERIVED PHARMACEUTICAL COMPOSITIONS |
US8021659B2 (en) * | 2006-04-28 | 2011-09-20 | Naidu Lp | Coenzyme Q10, lactoferrin and angiogenin compositions and uses thereof |
KR101193722B1 (en) * | 2006-07-24 | 2013-01-11 | 바이오렉시스 파마슈티칼 코포레이션 | Exendin fusion proteins |
ITBO20060891A1 (en) * | 2006-12-28 | 2008-06-29 | Cesare Pasquini | RECOMBINING YEAST LATTOFERRINA PIG, VECTOR OF EXPRESSION OF THE LATTOFERRINA SWINE GENE, RECOMBINING SWINE AND SUGAR PRODUCTION PROCESSES. |
NZ555163A (en) | 2007-05-14 | 2010-05-28 | Fonterra Co Operative Group | Methods of immune or hematological enhancement, inhibiting tumour formation or growth, and treating or preventing cancer, cancer symptoms, or the symptoms of cancer treatments |
JP2010533201A (en) | 2007-07-10 | 2010-10-21 | グランビア ニュートリショナルズ | Method for removing endotoxins from proteins |
EA013008B1 (en) | 2007-09-05 | 2010-02-26 | Николай Евгеньевич Шатуновский | Apolactoferrin compositions and methods of the use thereof for treating viral hepatitis c |
ITRM20080163A1 (en) * | 2008-03-26 | 2009-09-27 | Maurizio Acri | USE OF LATTOFERRINA FOR THE PREVENTION OF NEONATAL SEPSIS IN PREMATURED NEWBORNS |
MX348337B (en) * | 2010-11-26 | 2017-06-07 | Evonik Roehm Gmbh | Human lactoferrin derived peptide for use as an antigen masking agent. |
US20120171328A1 (en) * | 2011-01-05 | 2012-07-05 | Dattatreya Banavara | Composition comprising heat labile milk proteins and process for preparing same |
JP7370224B2 (en) * | 2019-11-08 | 2023-10-27 | ライオン株式会社 | Enteric-coated preparation containing lactoferrin |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3234199A (en) * | 1958-12-18 | 1966-02-08 | Allen F Reid | Ion exchange process for separating proteins |
US3969337A (en) * | 1973-04-18 | 1976-07-13 | Boehringer Mannheim G.M.B.H. | Chromatographic fractionation of whey |
US4436658A (en) * | 1981-05-15 | 1984-03-13 | Societe Nationale Elf Aquitaine | Process of extraction of lactoferrine and immunoglobulins of milk |
US4668771A (en) * | 1984-12-19 | 1987-05-26 | Snow Brand Milk Products Co., Ltd. | Method for separating bovine lactoferrin from cow's milk and purifying same |
US4732683A (en) * | 1986-12-02 | 1988-03-22 | Biospectrum, Inc. | Purification method for alpha interferon |
US5571896A (en) * | 1992-04-24 | 1996-11-05 | Baylor College Of Medicine | Production of recombinant human lactoferrin |
US5571697A (en) * | 1989-05-05 | 1996-11-05 | Baylor College Of Medicine Texas Medical Center | Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus |
US5571691A (en) * | 1989-05-05 | 1996-11-05 | Baylor College Of Medicine | Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL94183A (en) * | 1989-05-05 | 2003-09-17 | Baylor College Medicine | cDNA SEQUENCE CODING FOR HUMAN LACTOFERRIN PROTEIN OR PORTION THEREOF AND LACTOFERRIN PROTEIN PRODUCED FROM SAID SEQUENCE |
-
1996
- 1996-09-30 US US08/724,586 patent/US6066469A/en not_active Expired - Fee Related
-
1999
- 1999-10-19 US US09/421,632 patent/US6277817B1/en not_active Expired - Fee Related
-
2001
- 2001-08-17 US US09/932,190 patent/US6455687B1/en not_active Expired - Fee Related
- 2001-12-18 US US10/023,096 patent/US20020160941A1/en not_active Abandoned
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3234199A (en) * | 1958-12-18 | 1966-02-08 | Allen F Reid | Ion exchange process for separating proteins |
US3969337A (en) * | 1973-04-18 | 1976-07-13 | Boehringer Mannheim G.M.B.H. | Chromatographic fractionation of whey |
US4436658A (en) * | 1981-05-15 | 1984-03-13 | Societe Nationale Elf Aquitaine | Process of extraction of lactoferrine and immunoglobulins of milk |
US4668771A (en) * | 1984-12-19 | 1987-05-26 | Snow Brand Milk Products Co., Ltd. | Method for separating bovine lactoferrin from cow's milk and purifying same |
US4732683A (en) * | 1986-12-02 | 1988-03-22 | Biospectrum, Inc. | Purification method for alpha interferon |
US5571697A (en) * | 1989-05-05 | 1996-11-05 | Baylor College Of Medicine Texas Medical Center | Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus |
US5571691A (en) * | 1989-05-05 | 1996-11-05 | Baylor College Of Medicine | Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms |
US5571896A (en) * | 1992-04-24 | 1996-11-05 | Baylor College Of Medicine | Production of recombinant human lactoferrin |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1478379A2 (en) * | 2001-12-28 | 2004-11-24 | Campina B.V. | Humanized lactoferrin and uses thereof |
EP1478379A4 (en) * | 2001-12-28 | 2005-12-21 | Campina Bv | Humanized lactoferrin and uses thereof |
US20030229011A1 (en) * | 2001-12-28 | 2003-12-11 | Braun Steven O. | Humanized lactoferrin and uses thereof |
WO2005079582A1 (en) * | 2004-02-24 | 2005-09-01 | Campina B.V. | Antimicrobial lactoferrin compositions for surfaces, cavities, and foodstuff |
US20080318834A1 (en) * | 2004-02-24 | 2008-12-25 | Campina B.V. | Antimicrobial Lactoferrin Compositions for Surfaces, Cavities, and Foodstuff |
US20090306350A1 (en) * | 2005-05-13 | 2009-12-10 | Crea Biopharma Inc. | New purification method of lactoferrin |
EP1899375A1 (en) * | 2005-05-13 | 2008-03-19 | Array Biopharma Inc. | New purification method of lactoferrin |
EP1899375A4 (en) * | 2005-05-13 | 2009-05-06 | Crea Biopharma Inc | New purification method of lactoferrin |
US9149035B2 (en) | 2005-12-14 | 2015-10-06 | Convatec Technologies, Inc. | Antimicrobial composition |
US10493101B2 (en) | 2005-12-14 | 2019-12-03 | Convatec Technologies Inc. | Antimicrobial composition |
US20090291122A1 (en) * | 2006-06-01 | 2009-11-26 | Sano Medical Bvba | Wound Care Treatment Product |
WO2009102677A1 (en) * | 2008-02-11 | 2009-08-20 | Tabatchnick Fine Foods, Inc. | A nutritionally enhanced therapeutic preventative food supplement and method of making same |
US20100198946A1 (en) * | 2009-02-05 | 2010-08-05 | At&T Mobility Ii Llc | SYSTEM AND METHOD FOR QUALITY OF SERVICE (QoS) PARAMETER CHANGE |
US11135315B2 (en) | 2010-11-30 | 2021-10-05 | Convatec Technologies Inc. | Composition for detecting biofilms on viable tissues |
US20130323314A1 (en) * | 2011-12-30 | 2013-12-05 | University Of Hyderabad | Novel Nanoparticles of Lactoferrin Useful for Preparing a Pharmaceutical Composition Facilitating Easy Delivery of the Drug and a Process for Preparing the Same |
US11286601B2 (en) | 2012-12-20 | 2022-03-29 | Convatec Technologies, Inc. | Processing of chemically modified cellulosic fibres |
Also Published As
Publication number | Publication date |
---|---|
US6277817B1 (en) | 2001-08-21 |
US6455687B1 (en) | 2002-09-24 |
US6066469A (en) | 2000-05-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6277817B1 (en) | Human lactoferrin | |
US5087569A (en) | Antimicrobial proteins, compositions containing same and uses thereof | |
EP0272489B1 (en) | Antimicrobial proteins, compositions containing same and uses thereof | |
JP4317890B2 (en) | Bacterial production of hydrophobic polypeptides | |
US5571691A (en) | Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms | |
EP0503939B1 (en) | Antimicrobial peptide and antimicrobial agent | |
US20050064546A1 (en) | Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms | |
PH27060A (en) | Hybrid interferons their use as pharmaceutical compositions and as intermediate products for the preparation of anti-bodies and the use thereof and processes for preparing them | |
AU721954B2 (en) | Expression of lipoproteins | |
JPH09509175A (en) | Broad spectrum antimicrobial compounds and methods of use | |
JPH02500084A (en) | Bactericidal and/or bacteriostatic peptides, their isolation methods, their production and their applications | |
US5859223A (en) | Soluble CR1 derivatives | |
JPH07501447A (en) | Fusion protein containing MGF and IL-3 | |
US5849881A (en) | Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms | |
US6100054A (en) | Production for recombinant lactoferrin and lactoferrin polypeptides using DNA sequences in various organisms | |
EP0629213B1 (en) | Use of an immunostimulatory agent | |
US5766939A (en) | Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms | |
Couto et al. | eNAP-2, a novel cysteine-rich bactericidal peptide from equine leukocytes | |
JPH09512423A (en) | Α-1-antichymotrypsin analog having elastase inhibitory activity | |
WO1991013982A1 (en) | Genetically engineered human lactoferrin | |
JP2012500257A (en) | Novel use of bacteriocin derived from Enterococcus faecalis SL-5 | |
WO1995030339A1 (en) | Cloning, expression, and uses of human lactoferrin | |
US6015889A (en) | Protein rib, a cell surface protein that confers immunity to many strains of the group B streptococcus: process for purification of the protein, reagent kit and pharmaceutical composition | |
JP3127298B2 (en) | Methods for isolating and expressing genes encoding streptokinase, resulting nucleotide sequences, recombinant DNA and transformed microorganisms | |
KR102314731B1 (en) | Recombinant endolysin ClyC with potential bacteriocidal activity against Staphylococcus aureus |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- AFTER EXAMINER'S ANSWER OR BOARD OF APPEALS DECISION |