WO2010050439A1 - タンパク質固定化用担体およびその利用 - Google Patents

タンパク質固定化用担体およびその利用 Download PDF

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WO2010050439A1
WO2010050439A1 PCT/JP2009/068345 JP2009068345W WO2010050439A1 WO 2010050439 A1 WO2010050439 A1 WO 2010050439A1 JP 2009068345 W JP2009068345 W JP 2009068345W WO 2010050439 A1 WO2010050439 A1 WO 2010050439A1
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nta
carrier
immobilized
protein
polyhistidine
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PCT/JP2009/068345
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English (en)
French (fr)
Japanese (ja)
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進司 桂
真彦 大重
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国立大学法人 群馬大学
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Publication of WO2010050439A1 publication Critical patent/WO2010050439A1/ja

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J45/00—Ion-exchange in which a complex or a chelate is formed; Use of material as complex or chelate forming ion-exchangers; Treatment of material for improving the complex or chelate forming ion-exchange properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6827—Total protein determination, e.g. albumin in urine
    • G01N33/683—Total protein determination, e.g. albumin in urine involving metal ions

Definitions

  • the present invention relates to a carrier capable of efficiently immobilizing or purifying a polyhistidine-containing protein, a protein immobilization kit or purification kit containing the carrier, and a protein immobilization method or purification method.
  • a polyhistidine tag is added to the amino terminus and expressed as a fusion protein, and the expressed protein is immobilized and purified by forming a chelate bond with a divalent metal ion.
  • Patent Document 1 In order to form a chelate bond with a divalent metal ion, a technique for directly binding nitrilotriacetic acid (NTA) onto a carrier such as a gel or a substrate has been developed.
  • NTA nitrilotriacetic acid
  • an object of the present invention is to provide a technique for increasing the amount of protein to be immobilized.
  • the present inventor has intensively studied to solve the above problems. As a result, by immobilizing chitosan on a carrier, immobilizing nitrilotriacetic acid (NTA) via the chitosan, and coordinating a divalent metal ion to the immobilized NTA, polyhistidine-containing protein is efficiently produced.
  • NTA nitrilotriacetic acid
  • the present invention has been completed by finding that it can be immobilized.
  • NTA-immobilized carrier containing chitosan immobilized on a carrier and nitrilotriacetic acid (NTA) bound to the chitosan, wherein a divalent metal ion is coordinated with NTA to produce a polyhistidine-containing protein.
  • NTA immobilization carrier that can be bound.
  • NTA was immobilized by reacting glutaraldehyde with the amino group of chitosan immobilized on a carrier and then reacting with N- (5-Amino-1-carboxypentyl) iminodiacetic acid.
  • a kit for purifying or immobilizing a polyhistidine-containing protein comprising the NTA-immobilized carrier according to any one of (1) to (3).
  • a protein chip comprising the NTA-immobilized carrier according to (3) and having a polyhistidine-containing protein bound to a plurality of locations on the NTA-immobilized carrier.
  • a divalent metal ion is coordinated to the NTA-immobilized carrier according to any one of (1) to (3), and then a polyhistidine-containing protein-containing sample is loaded on the carrier to thereby convert the polyhistidine-containing protein into the carrier.
  • a method for immobilizing a polyhistidine-containing protein comprising specifically binding to a polyhistidine.
  • a bivalent metal ion is coordinated to the NTA-immobilized carrier according to any one of (1) to (3), and then a polyhistidine-containing protein-containing sample is loaded on the carrier to thereby convert the polyhistidine-containing protein into the carrier.
  • (A) is immediately after His6-GFP spot
  • (B) is after washing with ultrapure water
  • (C) is after imidazole treatment.
  • Fluorescence image (photo) showing the binding of His6-GFP to chitosan-NTA modified substrate, glutaraldehyde-NTA modified substrate and polyallylamine-NTA modified substrate.
  • A) is immediately after His6-GFP spot
  • (B) is after washing with ultrapure water.
  • the fluorescence image (photograph) which shows the coupling
  • FIG. (A) is immediately after His6-GFP spot
  • (B) is after washing with ultrapure water. Fluorescence images (photos) showing the binding of His6-GFP on various substrates modified with chitosan-NTA.
  • the NTA-immobilized carrier of the present invention is an NTA-immobilized carrier containing chitosan immobilized on a carrier and nitrilotriacetic acid (NTA) bound to the chitosan, wherein a divalent metal ion is coordinated to NTA.
  • NTA nitrilotriacetic acid
  • An NTA-immobilized carrier capable of binding a polyhistidine-containing protein.
  • Examples of materials that can be used as the carrier include plastics, inorganic polymers, natural polymers, and ceramics.
  • plastics include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenolic resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyethylene fluoride, polyimide, and acrylic resin as inorganic polymers.
  • glass, quartz, carbon, silica gel, graphite, etc. natural polymers are cellulose, cellulose derivatives, alginic acid, alginates, etc.
  • ceramics are alumina, silica, silicon carbide, silicon nitride, boron carbide, etc. Etc. can be illustrated.
  • Examples of the shape of the carrier include films, flat plates, particles, molded products (beads, strips, multiwell plates, membranes, slides, cell culture containers, etc.), and latex. There is no limit.
  • Chitosan may be naturally derived or synthetic. If the amino group is intact to some extent for NTA immobilization (if conserved), it may be a modified form.
  • the molecular weight is not particularly limited, but high molecular chitosan having a molecular weight of 10,000 or more is preferable.
  • the method for immobilizing chitosan on the carrier is not particularly limited, but it is preferably bonded to the carboxyl group on the carrier via the amino group of chitosan.
  • the carrier is treated with aminosilane, the carrier is modified with an amino group, glutaraldehyde is reacted with the amino group to form a carboxyl group on the carrier, and this carboxyl group is reacted with a part of the amino group of chitosan.
  • the chitosan can be immobilized.
  • NTA can preferably be bound by reacting the remaining amino group of chitosan with glutaraldehyde to form a carboxyl group, which reacts with a compound containing NTA and containing an amino group. Examples of such a compound include N- (5-Amino-1-carboxypentyl) iminodiacetic acid (AB-NTA).
  • the glass is modified in the following order: aminosilane, glutaraldehyde, chitosan, glutaraldehyde, AB-NTA. Chitosan modification may be performed twice or more. As a result, the amount of immobilization is increased, and uneven immobilization can be reduced.
  • it is immersed in 1M sodium hydroxide and treated for 3 days, then washed with ultrapure water three times with acetone and ethanol, and air-dried.
  • the amino group of chitosan is covalently bonded to the aldehyde group of glutaraldehyde, and the surface area is increased by modification with chitosan, which is a polysaccharide rich in amino groups, resulting in a large number of amino groups on the substrate.
  • the sample is again immersed in a glutaraldehyde solution adjusted to pH 7 with buffer HEPES, treated overnight, then washed with ultrapure water three times and ethanol once and air dried.
  • about 150 mM AB-NTA adjusted to pH 8 with HEPES is applied on a glass substrate, and a parafilm is placed on the glass substrate and allowed to react overnight.
  • the aldehyde group and the amino group of NTA are bonded, and NTA is fixed on the glass substrate via chitosan.
  • the type of the substrate is not limited to the glass substrate, and the substrate can be fixed to a substrate made of a material such as a silicon wafer, glassy carbon, polycarbonate, or metal.
  • the divalent metal ion can be coordinated with NTA.
  • examples of the divalent metal ion include Cu 2+ , Zn 2+ , Ni 2+ , Ca 2+ , Co 2+ , and Mg 2+ , and Ni 2+ is more preferable.
  • These metal ions can be added as a metal salt solution (eg, NiCl 2 solution).
  • the NTA-immobilized carrier of the present invention in which a divalent metal ion is coordinated can be used for binding or purification of a polyhistidine-containing protein.
  • polyhistidine refers to a polypeptide site in which two or more histidines are continuous.
  • the number of consecutive histidines may be in the range that does not affect the function of the protein immobilized via the polyhistidine site, but is preferably 6 as long as it is 2 or more.
  • Polyhistidine may be introduced at any site of the protein to be immobilized, but the amino terminus or carboxy terminus is preferred.
  • the protein to be immobilized is not particularly limited, and examples thereof include enzymes, antibodies, peptides, transcription factors, receptors, viral antigens, fluorescent proteins, and protein A. Moreover, you may use a random protein as a protein library.
  • the protein to be immobilized may be a non-natural protein that is chemically synthesized.
  • the protein to be immobilized in the present invention includes peptides.
  • the protein immobilized on the protein chip of the present invention can be expressed, for example, by expressing a vector containing the DNA encoding the protein in vitro such as reticulocyte lysate or in host cells such as E. coli or insect cells. Obtainable. It can also be obtained by chemical synthesis.
  • a protein containing polyhistidine which is a protein suitable for the present invention includes, for example, a known polyhistidine fusion protein expression vector containing a DNA encoding a target protein (including a sequence encoding polyhistidine, and a fusion protein with polyhistidine is A vector for incorporating a gene of a target protein so as to be expressed, for example, by incorporating into a pET series (Novagen) and transforming Escherichia coli with the vector to express a fusion protein.
  • an oligonucleotide containing a sequence encoding polyhistidine and its complementary strand are synthesized, both strands are hybridized, and incorporated into an expression vector containing DNA encoding the target protein so that the fusion protein is expressed. It can also be obtained by expressing a fusion protein by transforming E. coli or the like with
  • the resulting fraction containing the polyhistidine-containing protein can be bound to the NTA-immobilized carrier by incubating with an NTA-immobilized carrier coordinated with a divalent metal ion and washing.
  • the obtained polyhistidine-containing protein binding carrier can also be used as a protein chip.
  • an enzyme it can also be used in an assay for an inhibitor of the enzyme.
  • an antibody it can also be used for ELISA or antigen purification.
  • a substrate having a plurality of chitosan-NTA immobilized on the substrate and binding proteins thereto it can be used as a protein array in a multi-assay or the like.
  • the polyhistidine-containing protein can be purified by binding the polyhistidine-containing protein to the NTA-immobilized carrier as described above, washing, and then eluting with imidazole or the like. Since the amount of protein that can be bound can be increased by using the carrier of the present invention, purification can be performed efficiently.
  • the kit of the present invention is a kit for purifying or immobilizing a polyhistidine-containing protein containing the NTA immobilization carrier.
  • the kit of the present invention may further contain a divalent metal ion solution, a polyhistidine-containing protein expression vector, imidazole, and the like.
  • Example 1 An NTA-immobilized substrate was prepared by the following procedure. In addition, the board
  • Glutaraldehyde treatment ⁇ br/> Glutaraldehyde solution (100 mM HEPES pH7, 2.5 v / v% glutaraldehyde) 100 ml, 4 glass substrates and let stand overnight. ⁇ Ultrapure water x 3, washed with ethanol, air dried. Save one for comparison at the time of GFP spot.
  • Chitosan treatment ⁇ br/> Chitosan solution (100 mM HEPES pH8, Chitosan (SIGMA Cat. No.3646, 85% or more deacetylated) 0.5%) 3 glass substrates are placed in a vat and stirred with a stirrer While standing overnight.
  • ⁇ Glutaraldehyde treatment Make 80ml of glutaraldehyde solution (100mM HEPES pH7, 2.5v / v% glutaraldehyde), put two glass substrates and let stand overnight with stirring in a vat. ⁇ Ultrapure water x 3, washed with ethanol, air dried. Save one for comparison at the time of GFP spot.
  • ⁇ AB-NTA treatment Add AB-NTA (DOJINDO Cat. No.
  • GFP spot 6 substrates after each step (after NaOH cleaning, after aminosilane treatment, after glutaraldehyde treatment 1, after chitosan treatment, after glutaraldehyde treatment 2, after AB-NTA treatment) Spot 4 ⁇ l of GFP (Green fluorescent protein (His6 added): 3.0 mg / ml) in 1 well, and confirm fluorescence with a transilluminator.
  • GFP Green fluorescent protein (His6 added): 3.0 mg / ml
  • FIG. 1A shows the fluorescence when spotted on each substrate. At the time of spotting, fluorescence of GFP was seen on any substrate. In the Ni-NTA modified substrate (right), the spotted GFP spread on the substrate, so that the fluorescence appears thin.
  • FIG. 1B shows the fluorescence after washing with ultrapure water. Adsorption of GFP can be confirmed on the Ni-NTA modified substrate. In other cases, GFP flowed out by washing with ultrapure water.
  • FIG. 1C shows the fluorescence after imidazole treatment. Most of the GFP immobilized on the Ni-NTA modified substrate was eluted with imidazole. From this, it was confirmed that the His-tagged GFP (His6-GFP) protein was immobilized by Ni-NTA.
  • Example 2 In addition to the chitosan-NTA modified glass substrate prepared above, His6-GFP was also spotted on the following two NTA modified glass substrates for comparison.
  • Glutaraldehyde-NTA modified substrate AB-NTA modification after the first glutaraldehyde treatment.
  • Polyallylamine (PAA) -NTA modified substrate After the first treatment with glutaraldehyde, soaked in a PAA (polyamine) solution (100 mM HEPES pH 8 0.15% PAA) and then modified with glutaraldehyde and AB-NTA.
  • PAA polyamine
  • the surface area is increased by modifying the glass substrate with chitosan, and the modification efficiency of Ni-NTA is increased because many amino groups on the chitosan molecule and AB-NTA are cross-linked by glutaraldehyde. Immobilization of His tag fusion protein became possible. In addition, since the bond between the nickel complex and the His tag is used, the protein is oriented and the protein can be immobilized while maintaining the activity. By increasing the amount of protein immobilized on the substrate, a protein array effective for detecting the interaction can be created. In fact, the amount of GFP, which was so small that fluorescence could not be detected by the conventional method, was adsorbed on the substrate on the transilluminator.
  • Example 3 An NTA-immobilized substrate was prepared by the following procedure (description of the cleaning process omitted). Glass substrate cleaning (1M NaOH) A masked, unmodified glass substrate (MATSUNAMI SLIDE GLASS ring mark) is immersed in 1M NaOH for a day. ⁇ Aminosilane treatment ⁇ br/> Aminosilane (1% 3-Aminopropyltriethoxysilane) and a glass substrate were placed in a desiccator and decompressed, and left for 2 hours. ⁇ Bake glass substrate at 100 ° C for 2 hours.
  • Glutaraldehyde treatment A glass substrate is attached to a glutaraldehyde solution (100 mM HEPES pH 7, 0.5 v / v% glutaraldehyde) and left at 37 ° C. with stirring for 3 days.
  • a Chitosan treatment ⁇ br/> A glass substrate is attached to a chitosan solution (100 mM HEPES pH 8, chitosan (SIGMA Cat. No. 3646, 85% or more deacetylated) 0.05%) and left at 37 ° C with stirring for one day.
  • ⁇ Glutaraldehyde treatment A glass substrate is attached to a glutaraldehyde solution (100 mM HEPES pH 7, 0.5 v / v% glutaraldehyde) and left at 37 ° C. with stirring for one day.
  • ⁇ AB-NTA treatment AB-NTA solution 300 mM HEPES pH 8, 130 mM AB-NTA concentration
  • NiCl 2 treatment Spot a 1.35M NiCl 2 aqueous solution, put parafilm from the top, and let stand for 1 hour.
  • ⁇ GFP spot Spot GFP His6 addition: 2.08 mg / ml), let stand for 1 hour, wash with ultrapure water, and confirm fluorescence with a transilluminator.
  • Example 4 An NTA-immobilized substrate was prepared by the following procedure (description of the cleaning process omitted). Glass substrate cleaning (1M NaOH) A masked, unmodified glass substrate (MATSUNAMI SLIDE GLASS ring mark) is immersed in 1M NaOH for a day. ⁇ Aminosilane treatment ⁇ br/> Aminosilane (1% 3-Aminopropyltriethoxysilane) and a glass substrate were placed in a desiccator and decompressed, and left for 2 hours. ⁇ Bake glass substrate at 100 ° C for 2 hours.
  • Glutaraldehyde treatment A glass substrate is attached to a glutaraldehyde solution (100 mM HEPES pH 7, 0.5 v / v% glutaraldehyde) and left at 37 ° C. with stirring for 3 days.
  • a Chitosan treatment ⁇ br/> A glass substrate is attached to a chitosan solution (100 mM HEPES pH 8, chitosan (SIGMA Cat. No. 3646, 85% or more deacetylated) 0.05%) and left at 37 ° C with stirring for one day.
  • Glutaraldehyde treatment A glass substrate is attached to a glutaraldehyde solution (100 mM HEPES pH 7, 0.5 v / v% glutaraldehyde) and left at 37 ° C. with stirring for one day.
  • a Chitosan treatment ⁇ br/> A glass substrate is attached to a chitosan solution (100 mM HEPES pH 8, chitosan (SIGMA Cat. No. 3646, 85% or more deacetylated) 0.05%) and left at 37 ° C with stirring for one day.
  • ⁇ Glutaraldehyde treatment A glass substrate is attached to a glutaraldehyde solution (100 mM HEPES pH 7, 0.5 v / v% glutaraldehyde) and left at 37 ° C. with stirring for one day.
  • ⁇ AB-NTA treatment AB-NTA solution 300mM HEPES pH8) (AB-NTA concentration 130mM) is spotted on a glass substrate at four locations, left on top in a tapper with parafilm on top.
  • ⁇ NiCl 2 treatment Spot a 1.35M NiCl 2 aqueous solution, put parafilm from the top, and let stand for 1 hour.
  • ⁇ GFP spot Spot GFP His6 addition: 2.08 mg / ml), let stand for 1 hour, wash with ultrapure water, and confirm fluorescence with a transilluminator.
  • ⁇ Glutaraldehyde treatment A glass substrate is attached to a glutaraldehyde solution (100 mM HEPES pH 7, 0.5 v / v% glutaraldehyde) and left at 37 ° C. with stirring for 3 days.
  • ⁇ Branched polyethyleneimine (PEIb) treatment Place a glass substrate in PEIb solution (100 mM HEPES pH 8, branched chain polyethyleneimine 0.5%) and leave it at 37 ° C for 1 day.
  • PEIb Polyethyleneimine
  • PEIb Polyethyleneimine
  • PEIb solution 100 mM HEPES pH 8, branched chain polyethyleneimine 0.5%) and leave it at 37 ° C for 1 day.
  • Glutaraldehyde treatment A glass substrate is attached to a glutaraldehyde solution (100 mM HEPES pH 7, 0.5 v / v% glutaraldehyde) and left at 37 ° C. with stirring for one day.
  • AB-NTA treatment AB-NTA solution (300mM HEPES pH8) (AB-NTA concentration 130mM) is spotted on a glass substrate at four locations, left on top in a tapper with parafilm on top.
  • ⁇ GFP spot Spot GFP His6 addition: 2.08 mg / ml, let stand for 1 hour, wash with ultrapure water, and confirm fluorescence with a transilluminator.
  • Example 5 Chitosan-NTA modification is applied to each substrate made of silicon wafer, glassy carbon, polycarbonate and gold foil, and NiCl 2 aqueous solution is spotted on each obtained chitosan-NTA modified substrate, and then His6-GFP is bound. I let you.
  • the silicon wafer substrate was modified with chitosan-NTA in the same procedure as in Example 3. Further, the glassy carbon substrate was polished with sandpaper, washed with ultrapure water and ethanol, respectively, and then subjected to chitosan-NTA modification by the procedure after the aminosilane treatment in Example 3.
  • the polycarbonate substrate was washed with ultrapure water and ethanol, respectively, and then subjected to the chitosan-NTA modification by performing the procedure after the aminosilane treatment in Example 3.
  • a cysteine solution (0.67 M L-cysteine, 0.5 ⁇ TBE, 10 mM NaCl) was spotted instead of aminosilane deposition, incubated for 2 hours, washed with ultrapure water, and then Example 3
  • the chitosan-NTA modification was performed by performing the procedure after treatment with glutaraldehyde.
  • FIG. 4 shows the results of spotting His6-GFP and observing with a fluorescence microscope after washing. As a result, it was found that His6-GFP was efficiently bound to any substrate via chitosan-NTA.
  • the present invention it is possible to increase the amount of protein immobilized on a carrier such as a glass substrate by chitosan, thereby making it possible to create a protein array effective for detecting protein interactions.
  • a large amount of protein can be purified.
PCT/JP2009/068345 2008-10-27 2009-10-26 タンパク質固定化用担体およびその利用 WO2010050439A1 (ja)

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WO2012030961A3 (en) * 2010-08-31 2012-07-05 Massachusetts Institute Of Technology A nanotube array for optical detection of protein-protein interactions
KR20160056677A (ko) * 2014-11-12 2016-05-20 한국과학기술연구원 인 제거용 키토산 복합체 및 이의 제조방법
JP2016534373A (ja) * 2013-10-09 2016-11-04 ピュリディファイ リミテッド クロマトグラフィー担体
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KR101672231B1 (ko) 2014-11-12 2016-11-04 한국과학기술연구원 인 제거용 키토산 복합체 및 이의 제조방법
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