WO1991013982A1 - Genetically engineered human lactoferrin - Google Patents

Genetically engineered human lactoferrin Download PDF

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Publication number
WO1991013982A1
WO1991013982A1 PCT/US1991/001335 US9101335W WO9113982A1 WO 1991013982 A1 WO1991013982 A1 WO 1991013982A1 US 9101335 W US9101335 W US 9101335W WO 9113982 A1 WO9113982 A1 WO 9113982A1
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Prior art keywords
lactoferrin
adsorbed
adsorbent
column
human lactoferrin
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PCT/US1991/001335
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French (fr)
Inventor
Marian L. Kruzel
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Ferrodynamics, Inc.
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Publication of WO1991013982A1 publication Critical patent/WO1991013982A1/en

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    • 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
    • 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
    • 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 to human lactoferrin.
  • human 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.
  • the binding constant of iron by lactoferri •n is about 1020 M-1. Although lactoferri•n binds iron tightly, the binding is reversible so that the metal is available upon demand to cells with a need for this essential element.
  • 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.
  • production of lactoferrin from human milk poses problems. First, the severely limited amount of human milk, the major source of human lactoferrin, that is available restricts lactoferrin production. Secondly, production of lactoferrin from human milk presents a tremendous risk factor of infectious contamination. That is, it could carry with it a potentially lethal contaminant, such as the AIDS virus, or another undesirable agent.
  • the present invention provides human lactoferrin expressed from recombinant DNA.
  • the lactoferrin of the present invention is free of naturally occurring contaminants, e.g., proteins and viruses, that would be detrimental to the recipient.
  • the present invention also provides a genetically altered organism capable of producing human lactoferrin.
  • the present invention provides a method of producing human lactoferrin comprising the steps of (a) isolating DNA encoding human lactoferrin from a cDNA library derived from human breast tissue;
  • the present invention also provides a nutritional supplement comprising the trace-element loaded human lactoferrin having at least 35% metal loading and a nutritionally acceptable carrier or diluent.
  • the present invention further provides a topical antiseptic comprising an effective amount of lactoferrin having less than 25% metal loading and a pharmaceutically acceptable carrier or diluent.
  • Still another aspect of the present invention provides a process for inhibiting food spoilage comprising adding to the food an effective amount of lactoferrin having less than 25% metal loading.
  • the present invention also contemplates an improvement in a chromatography process useful in purifying lactoferrin and other proteins comprising the steps of (a) contacting a substance with a first adsorbent to obtain adsorbed and non- adsorbed 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.
  • Fig. 1 is a schematic diagram showing a preferred embodiment of the improved chromatography method of the present invention.
  • Fig. 2 is a flow chart showing a preferred embodiment of purifying lactoferrin in accordance with the present invention.
  • Lactoferrin is produced according to the present invention using recombinant DNA technology. That is, by using recombinant DNA technology, a polypeptide containing the primary structural conformation of naturally occurring human lactoferrin and possessing its biological properties is produced.
  • ⁇ is a cDNA library derived from human RNA and ligated to an appropriate vector according to methods that will be readily apparent to the skilled artisan, e.g., as disclosed in Davis, et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc, (1986), the disclosure of which is incorporated herein by reference.
  • the RNA is isolated from the human mammary gland and the vector is phage gt 11.
  • the cDNA library is screened for positive (DNA carrying the lactoferrin gene) clones using techniques that will be readily apparent to the skilled artisan, such as disclosed in the aforesaid Davis, et al., publication and Rado, et al., Blood, 70, No.
  • the cDNA is hybridized to a radiolabeled oligonucleotide probe and the positive clones identified by autoradiography.
  • positive clones are identified using lactoferrin antisera, and the antisera- containing clones are recognized using 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 disclosed in the aforesaid Davis, et al., publication. DNA is then isolated from the clones.
  • the cDNA encoding lactoferrin is then 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.
  • human lactoferrin is performed using an appropriate expression vector, such as the plasmid pAO804, and an appropriate host organism, such as the yeast Pichia pastoris.
  • an appropriate expression vector such as the plasmid pAO804, and an appropriate host organism, such as the yeast Pichia pastoris.
  • Other useful hosts include a mammalian cell line such as Chinese hamster ovary (CHO) and expression vectors that will be readily apparent to the skilled artisan. Insertion of the cDNA and expression of the human lactoferrin are carried out according to techniques that will be readily apparent to the skilled artisan, such as disclosed in Rothstein, Methods in Enzymolocry, 101, 202-210 (1983), and Tschopp, et al. , Bio/Technology, 5, 1305-1308 (1987) , the disclosures of which are incorporated herein by reference.
  • Purification of the expressed protein according to the present invention is preferably carried out by one of several methods.
  • cell-free culture media containing the expressed lactoferrin 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)nm) disclosed in Bezwoda, et al., Clin. Chim. Acta. , 157, 89-94 (1986) , and Chemical Abstracts Service (CAS) No.
  • Cibacron blue F3G-A can be covalently bound to a cross-linked agarose gel by the triazine coupling method as described in Boh e, et al., J. Chromatography, 69. 209- 214 (1972) , the disclosure of which is incorporated herein by reference.
  • controlled-pore glass (CPG) or silicic acid is used to further purify the adsorbed material obtained in the first step.
  • 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 ultrafiltrate 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.
  • 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 not shown
  • the nutritional supplement of the present invention contains an effective amount of lactoferrin loaded with one or more trace elements, either alone or in combination with one or more nutritionally acceptable carriers or diluents.
  • Preferred nutritional supplements include tablets, gelatin capsules, or liquids containing the lactoferrin together with 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.
  • diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine
  • the trace-element 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 trace element-loaded lactoferrin in the supplement based on the weight of lg of the supplement is 5-50 mg, more preferably 20-30 mg, most preferably 25 mg.
  • An effective daily amount of trace element-loaded lactoferrin varies, based on the individual, from about 10-30 mg, preferably 20- 30 mg, more preferably 25 mg.
  • the preferred trace element in the supplement is iron, but other trace elements, e.g., zinc and copper, are contemplated within the scope of the present invention.
  • Loading lactoferrin with the appropriate trace element 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 35%, more preferably at least 50%, most preferably at least 70%, of the metal-binding sites are bound to the trace element.
  • the lactoferrin contemplated for use in the supplement is preferably of human derivation, more preferably via DNA recombinant means, but other lactoferrins, such as bovine and porcine lactoferrin, are contemplated.
  • 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 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.
  • 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.
  • 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/6 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 same amounts of metal loading preferred for the antiseptic of the present invention are preferred for the food-spoilage retardant.
  • 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. To more clearly describe the present invention, the following non-limiting examples are provided. In the examples 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 (cDNA ligated to ⁇ gt 11) is prepared according to Gubler, et al.,
  • the library is transferred onto agar plates containing a high density of E. coli Y 1090 (available from Clontech, California) (5 x 10 4 plaques per 90 mm plate or 1.4 x 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.
  • E. coli Y 1090 available from Clontech, California
  • Positive clones i.e., containing the cDNA are identified on the membranes using rabbit antibody to natural human lactoferrin purified in accordance with Example 8 herein.
  • Nitrocellulose filters are removed from the plates after plaque transferral, and the coated with the antibody purified in accordance with Example 8, which hybridizes with positive plaques.
  • positive plaques are developed by first applying an anti-rabbit IgG conjugated with biotin (available from Sigma Chemical Co., St. Louis, MO) , and then, following removal of excess biotin conjugate, applying avidin conjugated with horse radish peroxidase (available from Sigma Chemical Co., St Louis, MO) .
  • the positive plaques are identified in the reaction catalyzed by horse radish peroxidase using as an enzyme substrate 4-chloro-l-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., 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.
  • the cDNA insert encoding lactoferrin is sub-cloned as follows: the cDNA insert is cut out from the phage DNA using EcoRI and purified using a high resolution ion-exchange chromatography column (Gen-PakTM Fax available from Millipore Corporation, Waters Chromatography Division, Milford, MA) . The thus purified cDNA insert is ligated using T4 DNA ligase into plasmid pGEM-4 (available from Promega, Madison, WI, and described in Yanish-Perron et al.
  • E. coli JM109 available from Promega, Madison, WI, and described in Hanahan, J. Mil. Biol. , 166, 557 (1983)). 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 EcoRI and purified by ion-exchange chromatography as described above.
  • the cDNA insert is then ligated into the Pichia pastoris expression vector pAO804 (using the P. pastoris GTS 115 strain) 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 in Sreekrishna, et al., Biochemistry, 28, 4117-4125 (1989), and Rothstein, Methods in Enzvmology, 101, 202-210 (1983) , the disclosures of which are incorporated herein by reference.
  • the thus modified vector is then grown in minimal media as described in Creeg, et al., Mol. Cell.
  • EXAMPLE 2 human lactoferrin is purified in accordance with the present invention.
  • One liter of the supernatant from Example 1 is adjusted to about 4°C 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 available from Millipore) to retain proteins in excess of about 10,000 molecular weight.
  • Pressure with simultaneous circulation is applied until 900 ml of ultrafiltrate is collected.
  • a flow rate of about 100 ml per minute is maintained during the filtration process.
  • the final material retained is sterilized (0.22 ⁇ m Gelman filter).
  • EXAMPLE 3 human lactoferrin is purified in accordance with a preferred embodiment of the present invention 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.
  • EXAMPLE 5 human lactoferrin is purified in accordance with a preferred embodiment of the present invention using immobilized metal ion affinity chromatography (IMAC) .
  • IMAC immobilized metal ion affinity chromatography
  • An imminodiacetic acid-epoxy activated gel (available from Pharmacia Fine Chemicals, Upsala, Sweden, under the name 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 x 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 imidazol.
  • Example 4 The product of Example 4 is mixed with HEPES, sodium chloride, and imidazol to obtain a pH of 7.0, 20 mM HEPES, 1 M sodium chloride, and 2 mM imidazol.
  • 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 imidazol.
  • 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 imidazol.
  • EXAMPLE 6 human lactoferrin is purified in accordance with a preferred embodiment of the present invention using T-Gel affinity chromatography.
  • T-gel adsorbent is prepared according to Porath, et al., Methods in Enzvmology, 44. 19- 45 (1976) , the disclosure of which is incorporated herein by reference, and packed into a column (1.2 cm x 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-ethanesulfonic acid] and 1,4- piperazinediethanesulfonic acid] 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 lml/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.
  • Sepharose TM CL-4B avai•lable from Pharmaci•a Fi•ne Chemi•cals, Upsala, 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 for use in accordance with a preferred embodiment of the present invention as described in Example 1 herein.
  • the adsorbent substrate for affinity chromatography is prepared by cyanogen bromide activation as described by Axen et al., Nature, 214, 1302-1304 (1967) .
  • the substrate available from Pharmacia Fine Chemicals, Upsala,
  • 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 (available from 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 steri •li•zed (0.22 ⁇ m GelmanTM fi•lter) and frozen at -20°C.

Abstract

Disclosed is human lactoferrin expressed from recombinant DNA, its method of production and purification and its use.

Description

GENETICALLY ENGINEERED HUMAN LACTOFERRIN
The present invention relates to human lactoferrin. In particular, it relates to human lactoferrin, its production, and its use. 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. The binding constant of iron by lactoferri •n is about 1020 M-1. Although lactoferri•n binds iron tightly, the binding is reversible so that the metal is available upon demand to cells with a need for this essential element.
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. However, production of lactoferrin from human milk poses problems. First, the severely limited amount of human milk, the major source of human lactoferrin, that is available restricts lactoferrin production. Secondly, production of lactoferrin from human milk presents a tremendous risk factor of infectious contamination. That is, it could carry with it a potentially lethal contaminant, such as the AIDS virus, or another undesirable agent.
Accordingly, the present invention provides human lactoferrin expressed from recombinant DNA. The lactoferrin of the present invention is free of naturally occurring contaminants, e.g., proteins and viruses, that would be detrimental to the recipient. The present invention also provides a genetically altered organism capable of producing human lactoferrin. In a further aspect, the present invention provides a method of producing human lactoferrin comprising the steps of (a) isolating DNA encoding human lactoferrin from a cDNA library derived from human breast tissue;
(b) inserting the isolated DNA into the DNA of a host organism; (c) culturing the host organism to express human lactoferrin; and (d) recovering the expressed lactoferrin from the culture media. In still another aspect of the present invention there is provided a process for inhibiting microbial growth in a mammal comprising topically applying to the subject a therapeutically effective amount of lactoferrin having less than 25% metal loading, a process for inhibiting a trace-element deficiency in a mammal comprising orally administering a therapeutically effective amount of lactoferrin having at least 35% trace-element loading. The present invention also provides a nutritional supplement comprising the trace-element loaded human lactoferrin having at least 35% metal loading and a nutritionally acceptable carrier or diluent. The present invention further provides a topical antiseptic comprising an effective amount of lactoferrin having less than 25% metal loading and a pharmaceutically acceptable carrier or diluent. Still another aspect of the present invention provides a process for inhibiting food spoilage comprising adding to the food an effective amount of lactoferrin having less than 25% metal loading. The present invention also contemplates an improvement in a chromatography process useful in purifying lactoferrin and other proteins comprising the steps of (a) contacting a substance with a first adsorbent to obtain adsorbed and non- adsorbed 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.
Fig. 1 is a schematic diagram showing a preferred embodiment of the improved chromatography method of the present invention. Fig. 2 is a flow chart showing a preferred embodiment of purifying lactoferrin in accordance with the present invention.
Lactoferrin is produced according to the present invention using recombinant DNA technology. That is, by using recombinant DNA technology, a polypeptide containing the primary structural conformation of naturally occurring human lactoferrin and possessing its biological properties is produced. The preferred source of DNA encoding lactoferrin
■ is a cDNA library derived from human RNA and ligated to an appropriate vector according to methods that will be readily apparent to the skilled artisan, e.g., as disclosed in Davis, et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc, (1986), the disclosure of which is incorporated herein by reference. In a preferred embodiment, the RNA is isolated from the human mammary gland and the vector is phage gt 11. The cDNA library is screened for positive (DNA carrying the lactoferrin gene) clones using techniques that will be readily apparent to the skilled artisan, such as disclosed in the aforesaid Davis, et al., publication and Rado, et al., Blood, 70, No. 4 (October, 1987) pp. 989-983, the disclosure of which is incorporated herein by reference. For example, the cDNA is hybridized to a radiolabeled oligonucleotide probe and the positive clones identified by autoradiography. Preferably, positive clones are identified using lactoferrin antisera, and the antisera- containing clones are recognized using 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 disclosed in the aforesaid Davis, et al., publication. DNA is then isolated from the clones. The cDNA encoding lactoferrin is then 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 expression vector, such as the plasmid pAO804, and an appropriate host organism, such as the yeast Pichia pastoris. Other useful hosts include a mammalian cell line such as Chinese hamster ovary (CHO) and expression vectors that will be readily apparent to the skilled artisan. Insertion of the cDNA and expression of the human lactoferrin are carried out according to techniques that will be readily apparent to the skilled artisan, such as disclosed in Rothstein, Methods in Enzymolocry, 101, 202-210 (1983), and Tschopp, et al. , Bio/Technology, 5, 1305-1308 (1987) , the disclosures of which are incorporated herein by reference.
Purification of the expressed protein according to the present invention is preferably carried out by one of several methods. In one preferred embodiment, cell-free culture media containing the expressed lactoferrin 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, χ max 605(374)nm) disclosed in Bezwoda, et al., Clin. Chim. Acta. , 157, 89-94 (1986) , and Chemical Abstracts Service (CAS) No. 12236-82-7, the disclosures of which are incorporated herein by reference. Cibacron blue F3G-A can be covalently bound to a cross-linked agarose gel by the triazine coupling method as described in Boh e, et al., J. Chromatography, 69. 209- 214 (1972) , the disclosure of which is incorporated herein 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, which can be coupled with an imminodiacetic acid-epoxy activated gel (IDA Me(II)) in accordance with Sulkowski, Frontiers in Bioprocessing, Sidkar et al., ed. , 343-353 (1990) , the disclosure of which is incorporated herein by reference; or chromatography with the ligand phenyl glycidyl ether, which can be coupled to a cross-linked agarose gel as disclosed in Janson and Riden, Protein Purification Principles High Resolution Methods and Applications, VSH Publishers New York (1989) , incorporated by reference herein. The two-step and three-step methods are schematically presented by the flow chart 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 to permeable membrane 3, which retains material having a molecular weight greater than 10,000 daltons and passes an ultrafiltrate 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. 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 nutritional supplement of the present invention contains an effective amount of lactoferrin loaded with one or more trace elements, either alone or in combination with one or more nutritionally acceptable carriers or diluents. Preferred nutritional supplements include tablets, gelatin capsules, or liquids containing the lactoferrin together with 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 trace-element 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 trace element-loaded lactoferrin in the supplement based on the weight of lg of the supplement is 5-50 mg, more preferably 20-30 mg, most preferably 25 mg. An effective daily amount of trace element-loaded lactoferrin varies, based on the individual, from about 10-30 mg, preferably 20- 30 mg, more preferably 25 mg. The preferred trace element in the supplement is iron, but other trace elements, e.g., zinc and copper, are contemplated within the scope of the present invention. Loading lactoferrin with the appropriate trace element 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 35%, more preferably at least 50%, most preferably at least 70%, of the metal-binding sites are bound to the trace element. The lactoferrin contemplated for use in the supplement is preferably of human derivation, more preferably via DNA recombinant means, but other lactoferrins, such as bovine and porcine lactoferrin, are contemplated. 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 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 lactoferrin per 0.1 square meter effected area is contemplated. For use as an antiseptic in accordance with the present invention the lactoferrin preferably has less than 25% of its metal-binding sites loaded, i.e., at least 75% of its metal-binding sites are available to sequester iron. More preferably, less than 20% of the metal-binding sites are loaded, most preferably less than 10%.
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/6 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 same amounts of metal loading preferred for the antiseptic of the present invention are preferred for the food-spoilage retardant.
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. To more clearly describe the present invention, the following non-limiting examples are provided. In the examples all parts and percentages are by weight unless indicated otherwise.
EXAMPLE 1 In this example, 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 (cDNA ligated to \ gt 11) is prepared according to Gubler, et al.,
Gen. , 40. 1-8 (1983) (available from Clontech, California) .
The library is transferred onto agar plates containing a high density of E. coli Y 1090 (available from Clontech, California) (5 x 104 plaques per 90 mm plate or 1.4 x 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 then overlaid with nitrocellulose filters
(available from Schleiher and Schnell Inc. ouburn, MA, 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 8 herein. Nitrocellulose filters are removed from the plates after plaque transferral, and the coated with the antibody purified in accordance with Example 8, which hybridizes with positive plaques. Following removal of excess antibody, positive plaques are developed by first applying an anti-rabbit IgG conjugated with biotin (available from Sigma Chemical Co., St. Louis, MO) , and then, following removal of excess biotin conjugate, applying avidin conjugated with horse radish peroxidase (available from 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 4-chloro-l-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., 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) , the disclosure of which is incorporated herein by reference.- Following the procedures in Davis, et al., supra, the cDNA insert encoding lactoferrin is sub-cloned as follows: the cDNA insert is cut out from the phage DNA using EcoRI and purified using a high resolution ion-exchange chromatography column (Gen-Pak™ Fax available from Millipore Corporation, Waters Chromatography Division, Milford, MA) . The thus purified cDNA insert is ligated using T4 DNA ligase into plasmid pGEM-4 (available from Promega, Madison, WI, and described in Yanish-Perron et al. (1985) , GEN, 33, 103-109) that has been cut using EcoRI using standard techniques. The plasmid containing the insert is then transferred into E. coli JM109 (available from Promega, Madison, WI, and described in Hanahan, J. Mil. Biol. , 166, 557 (1983)). 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 EcoRI and purified by ion-exchange chromatography as described above.
The cDNA insert is then ligated into the Pichia pastoris expression vector pAO804 (using the P. pastoris GTS 115 strain) 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 in Sreekrishna, et al., Biochemistry, 28, 4117-4125 (1989), and Rothstein, Methods in Enzvmology, 101, 202-210 (1983) , the disclosures of which are incorporated herein by reference. The thus modified vector is then grown in minimal media as described in Creeg, et al., Mol. Cell. Biol., 5, 3376-3385 (1985) , the disclosure of which is incorporated herein by reference. Following the procedure of Hagenson, et al., Enzyme Microb. Technol, 11, 650-656 (1989), the cells are grown to an OD600 of about 1.0 and then harvested, and washed with and suspended in minimal methanol media at an OD600 of 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' centrifugation at 5000 rpm using a Beckman J-21B with a Rotor JA 14.
EXAMPLE 2 In this example, human lactoferrin is purified in accordance with the present invention. One liter of the supernatant from Example 1 is adjusted to about 4°C 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 available from Millipore) to retain proteins in excess of about 10,000 molecular weight. Pressure with simultaneous circulation is applied until 900 ml of ultrafiltrate 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 μm Gelman filter).
EXAMPLE 3 In this example human lactoferrin is purified in accordance with a preferred embodiment of the present invention 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 x 35 cm) packed with cross- linked agarose coupled to the dye (available from Pharmacia Fine Chemicals, Upsala, Sweden, under the name Blue Sepharose CL-6B ) and previously equilibrated with 50 mM N-[2-hydroxyethyl]-piperazine- •-[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.
EXAMPLE 4 In this example human lactoferrin is purified in accordance with a preferred embodiment of the present invention using control pore glass (CPG) chromatography. The eluate from
Example 3 is applied onto a column (1.2 cm x 10 cm) packed
' with CPG beads (CPG 00350 available from Electro-Nucleonics, 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 wei •ght greater than 10,000 daltons (Ami•conTM YM 10). The fi «ltered materi•al i•s then steri•li•zed (0.22 μm GelmanTM fi•lter) and frozen at -20°C.
EXAMPLE 5 In this example human lactoferrin is purified in accordance with a preferred embodiment of the present invention using immobilized metal ion affinity chromatography (IMAC) . An imminodiacetic acid-epoxy activated gel (available from Pharmacia Fine Chemicals, Upsala, Sweden, under the name 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 x 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 imidazol.
The product of Example 4 is mixed with HEPES, sodium chloride, and imidazol to obtain a pH of 7.0, 20 mM HEPES, 1 M sodium chloride, and 2 mM imidazol. 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 imidazol. 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 imidazol.
EXAMPLE 6 In this example human lactoferrin is purified in accordance with a preferred embodiment of the present invention using T-Gel affinity chromatography. T-gel adsorbent is prepared according to Porath, et al., Methods in Enzvmology, 44. 19- 45 (1976) , the disclosure of which is incorporated herein by reference, and packed into a column (1.2 cm x 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-ethanesulfonic acid] and 1,4- piperazinediethanesulfonic acid] 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 lml/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.
EXAMPLE 7
In this example human lactoferrin is purified in accordance with a preferred embodiment of the present invention using a hydrophobic interaction chromatography on a cross-linked agarose gel coupled to phenyl glycidyl ether (Phenyl
Sepharose TM CL-4B avai•lable from Pharmaci•a Fi•ne Chemi•cals, Upsala, 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) . EXAMPLE 8
In this example anti-lactoferrin serum is purified by affinity chromatography for use in accordance with a preferred embodiment of the present invention as described in Example 1 herein. The adsorbent substrate for affinity chromatography is prepared by cyanogen bromide activation as described by Axen et al., Nature, 214, 1302-1304 (1967) . The substrate (available from Pharmacia Fine Chemicals, Upsala,
Sweden, under the name CNBr-Sepharose TM-4B) i•s coupled to human lactoferrin, which acts as the affinity ligand, as follows. One gram of substrate is swelled with 1 mM HC1 and washed with the same solvent on a sintered glass filter. Ten mg of natural human lactoferrin (available from Sigma Chemical Co., St. Louis, MO) is dissolved in 0.1 M NaHCO, 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 (available from 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 steri •li•zed (0.22 μm GelmanTM fi•lter) and frozen at -20°C.

Claims

Claimed is:
1. Human lactoferrin expressed by recombinant DNA.
2. Lactoferrin having less than 25% metal loading.
3. The lactoferrin of claim 2 that is human lactoferrin.
4. The lactoferrin of claim 2 having less than 10% metal loading.
5. Lactoferrin having at least 35% metal loading.
6. The lactoferrin of claim 5 having at least 50% metal loading.
7. The lactoferrin of claim 5 that is human lactoferrin.
8. A genetically altered organism capable of producing human lactoferrin.
9. The organism of claim 8 that is eukaryotic.
10. The organism of claim 8 that is Pichia pastoris.
11. A process for producing human lactoferrin comprising the steps of:
(a) isolating DNA encoding human lactoferrin from a cDNA library;
(b) inserting the isolated DNA into the DNA of a host organism;
(c) culturing the host organism to express human lactoferrin; and
(d) recovering the expressed human lactoferrin from culture media.
12. The process of claim 11 wherein the lactoferrin is recovered by the steps of (i) filtering the culture media to retain a first material having a molecular weight of at least 10,000 daltons, (ii) chromatographing the retained material on an adsorbent having chromophore C.I. 61211 as an affinity ligand to obtain adsorbed and non-adsorbed fractions, (ii) chromatographing the adsorbed fraction on a medium wherein the medium is controlled pore glass beads or silicic acid to obtain adsorbed and non-adsorbed fractions, and (iii) filtering the adsorbed fraction from the medium to retain a second material having a molecular weight of at least 10,000 daltons.
13. The process of claim 12 further comprising the steps of (iv) chromatographing the second retained material on T- Gel at least once to obtain adsorbed and non-adsorbed fractions, and (v) filtering the non-adsorbed fraction from the T-Gel to retain a third material having a molecular weight of at least 10,000.
14. The process of claim 12 further comprising the steps of (iv) chromatographing the second retained material on a third adsorbent having phenyl glycidyl ether as an affinity ligand to obtain adsorbed and non-adsorbed fractions, and (v) filtering the adsorbed fraction from the third adsorbent to retain a third material having a molecular weight of at least 10,000 daltons.
15. The process of claim 12 further comprising the steps of (iv) chromatographing the second retained material on a fourth adsorbent having an immobilized metal ion as an affinity ligand to obtain adsorbed and non-adsorbed fractions, and (v) filtering the adsorbed fraction to retain a fourth material having a molecular weight of at least 10,000 daltons.
16. A process for inhibiting microbial growth on a mammal comprising topically applying a therapeutically effective amount of lactoferrin having less than 25% metal loading.
17. The process of claim 16 wherein the lactoferrin is human lactoferrin expressed by recombinant DNA.
18. A process for inhibiting trace-element deficiency in a mammal comprising orally administering a nutritionally effective amount of lactoferrin having at least 35% trace- element loading.
19. The process of claim 18 wherein the lactoferrin is human lactoferrin expressed by recombinant DNA.
20. The process of claim 18 wherein the trace element is at least one of iron, copper, or zinc.
21. A method for retarding food-spoilage comprising applying to food an effective amount of lactoferrin having less than 25% metal loading.
22. The method of claim 21 wherein the lactoferrin is human lactoferrin expressed by recombinant DNA.
23. A nutritional supplement comprising an effective amount of lactoferrin having at least 35% trace-element loading and a nutritionally acceptable carrier or diluent.
24. The supplement of claim 23 wherein the lactoferrin is human lactoferrin expressed by recombinant DNA.
25. The supplement of claim 23 wherein the trace element is at least one of iron, copper, or zinc.
26. A disinfectant comprising an effective amount of lactoferrin having less than 25% metal loading and an acceptable carrier or diluent.
27. The disinfectant of claim 26 wherein the lactoferrin is human lactoferrin expressed by recombinant DNA.
28. A food-spoilage retardant comprising an effective amount of lactoferrin having less than 25% metal loading and an acceptable carrier or diluent.
29. The additive of claim 28 wherein the lactoferrin is human lactoferrin expressed by recombinant DNA.
30. In a chromatography process comprising the steps of (a) contacting a substance with a first adsorbent to obtain adsorbed and non-adsorbed fractions, (b) eluting the adsorbed fraction with an eluant, and (c) contacting the adsorbed fraction with a second adsorbent, the improvement comprising equilibrating the second adsorbent with the eluant followed by contacting the eluate containing the adsorbed fraction with the second adsorbent.
31. The process of claim 30 wherein the first adsorbent is contained in a first column connected to a second column containing the second adsorbent by a means through which the eluate passes from the first column to the second column.
32. The process of claim 31 wherein the means sequentially (i) discharges material not adsorbed on the first adsorbent from the first column and (ii) passes the eluate from the first column to the second column.
33. The process of claim 32 further comprising the sequential steps of (d) discharging non-adsorbed material from the second-adsorbent, (e) eluting adsorbed material from the second adsorbent, and (f) filtering the eluate, wherein the non-adsorbed material and the eluate pass through a means capable of directing the effluent from the second column to the filtering means or the discharge locus.
34. The process of claim 31 further comprising the steps of obtaining the substance by passing a mixture through a filter that retains the substance, and prior to contacting the substance with the first adsorbent, washing the substance on the filter with a buffer and equilibrating the first adsorbent with the buffer.
35. A chromatography apparatus comprising (a) a first column containing a first adsorbent, (b) a second column containing a second adsorbent, and (c) a means capable of (i) carrying effluent from the first column to the second column or (ii) discharging the effluent.
36. The apparatus of claim 35 further comprising (d) means capable of directing effluent from the second column to at least two alternative loci.
37. In a chromatography process comprising the steps of (a) equilibrating an adsorbent with a buffer and (b) applying a protein-containing material to the adsorbent to obtain adsorbed and non-adsorbed fractions, the improvement comprising passing the material through a filter capable of excluding material having a molecular weight greater than 10,000 daltons and washing the material on the filter with the buffer before applying the material to the adsorbent.
38. A process for producing human lactoferrin comprising the steps of:
(a) culturing a host cell genetically altered to carry DNA encoding human lactoferrin to express human lactoferrin and
(b) recovering the expressed lactoferrin from culture media.
39. Genetically modified DNA capable of expressing human lactoferrin in a host organism.
PCT/US1991/001335 1990-03-08 1991-02-28 Genetically engineered human lactoferrin WO1991013982A1 (en)

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EP0603187A1 (en) * 1991-05-31 1994-06-29 THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by the SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES Human lactoferrin
EP0644899A1 (en) * 1992-04-24 1995-03-29 Baylor College Of Medicine Production of recombinant human lactoferrin
WO1995030339A1 (en) * 1994-05-05 1995-11-16 Ferrodynamics, Inc. Cloning, expression, and uses of human lactoferrin
WO1996014413A1 (en) * 1994-11-02 1996-05-17 Agennix, Inc. 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
WO1997045136A1 (en) * 1996-05-31 1997-12-04 Baylor College Of Medicine Lactoferrin variants and uses thereof
WO2000004132A1 (en) * 1998-07-15 2000-01-27 Samyang Genex Corporation Mass production method of lactoferrin polypeptide from yeast and useful microorganism thereof
WO2000072874A1 (en) * 1999-05-28 2000-12-07 Naidu A Satyanarayan IMMOBILIZED LACTOFERRIN (Im-LF) ANTIMICROBIAL AGENTS AND USES THEREOF
US6228614B1 (en) 1989-05-05 2001-05-08 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms
US6423509B1 (en) 1998-07-15 2002-07-23 Samyang Genex Corporation Pichia pastoris strain for producing lactoferrin and methods of use
US7034126B2 (en) * 2003-05-14 2006-04-25 Agennix, Inc. Lactoferrin in the treatment of diabetes mellitus
US7125963B2 (en) 2004-03-03 2006-10-24 En N Tech Inc Treatments for contaminant reduction in lactoferrin preparations and lactoferrin containing compositions
US7183381B2 (en) 2004-10-26 2007-02-27 Agennix, Inc. Composition of lactoferrin related peptides and uses thereof
WO2023004153A1 (en) * 2021-07-23 2023-01-26 Clara Foods Co. Purified protein compositions and methods of production

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US5955316A (en) * 1989-05-05 1999-09-21 Agennix, Inc. Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in aspergillus
US6228614B1 (en) 1989-05-05 2001-05-08 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms
US6080559A (en) * 1989-05-05 2000-06-27 Agennix, Inc. 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
EP0603187A1 (en) * 1991-05-31 1994-06-29 THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by the SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES Human lactoferrin
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US5948613A (en) * 1991-05-31 1999-09-07 The United States Of America As Represented By The Department Of Health And Human Services Methods of screening for risk of cancer using human lactoferrin DNA probe or primer
US5571896A (en) * 1992-04-24 1996-11-05 Baylor College Of Medicine Production of recombinant human lactoferrin
EP0644899A4 (en) * 1992-04-24 1996-06-26 Baylor College Medicine Production of recombinant human lactoferrin.
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WO1995030339A1 (en) * 1994-05-05 1995-11-16 Ferrodynamics, Inc. Cloning, expression, and uses of human lactoferrin
WO1996014413A1 (en) * 1994-11-02 1996-05-17 Agennix, Inc. Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in aspergillus
US6111081A (en) * 1996-05-31 2000-08-29 Baylor College Of Medicine Lactoferrin variants and uses thereof
WO1997045136A1 (en) * 1996-05-31 1997-12-04 Baylor College Of Medicine Lactoferrin variants and uses thereof
US6423509B1 (en) 1998-07-15 2002-07-23 Samyang Genex Corporation Pichia pastoris strain for producing lactoferrin and methods of use
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US7375080B1 (en) * 1999-05-28 2008-05-20 Naidu A Satyanarayan Immobilized lactoferrin (Im-LF) antimicrobial agents and uses thereof
WO2000072874A1 (en) * 1999-05-28 2000-12-07 Naidu A Satyanarayan IMMOBILIZED LACTOFERRIN (Im-LF) ANTIMICROBIAL AGENTS AND USES THEREOF
WO2000072690A3 (en) * 1999-05-28 2001-05-10 A Satyanarayan Naidu IMMOBILIZED LACTOFERRIN (Im-LF) ANTIMICROBIAL AGENTS AND USES THEREOF
AU776657B2 (en) * 1999-05-28 2004-09-16 A. Satyanarayan Naidu Immobilized lactoferrin (Im-LF) antimicrobial agents and uses thereof
US7034126B2 (en) * 2003-05-14 2006-04-25 Agennix, Inc. Lactoferrin in the treatment of diabetes mellitus
US7326775B2 (en) 2004-03-03 2008-02-05 En-N-Tech, Inc. Treatments for contaminant reduction in lactoferrin preparations and lactoferrin-containing compositions
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US7183381B2 (en) 2004-10-26 2007-02-27 Agennix, Inc. Composition of lactoferrin related peptides and uses thereof
US7420033B2 (en) 2004-10-26 2008-09-02 Agennix, Inc. Composition of lactoferrin related peptides and uses thereof
WO2023004153A1 (en) * 2021-07-23 2023-01-26 Clara Foods Co. Purified protein compositions and methods of production
US11718644B2 (en) 2021-07-23 2023-08-08 Clara Foods Co. Purified protein compositions and methods of production

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