JPWO2010050439A1 - Protein immobilization carrier and use thereof - Google Patents

Abstract

An NTA-immobilized carrier comprising chitosan immobilized on a carrier and nitrilotriacetic acid (NTA) bound to the chitosan, wherein a divalent metal ion is coordinated to NTA to bind a polyhistidine-containing protein. NTA immobilization carrier that can be used.

Description

  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.

  To efficiently immobilize and purify the recombinant protein, 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. Is known (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. As a method for preparing a protein array, a method has been developed in which a polyhistidine-containing protein is expressed on a DNA array and immobilized on a glass substrate directly modified with Ni-NTA (transcribed protein on the array). array). However, these methods have a problem in detection sensitivity because the amount of the immobilized protein is not sufficient.

Japanese Patent Laid-Open No. 2001-083155

  In the production of a protein array by a conventional transcription type, the amount of protein immobilized is not sufficient for detection. Therefore, 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. The present invention has been completed by finding that it can be immobilized.

That is, the present invention is as follows.
(1) 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 with NTA to produce a polyhistidine-containing protein. NTA immobilization carrier that can be bound.
(2) 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. 1) NTA-immobilized carrier.
(3) The NTA-immobilized carrier according to (1) or (2), wherein the carrier is a substrate, and chitosan-NTA is immobilized at a plurality of locations on the substrate.
(4) A kit for purifying or immobilizing a polyhistidine-containing protein comprising the NTA-immobilized carrier according to any one of (1) to (3).
(5) The kit according to (4), further comprising a divalent metal ion solution.
(6) The kit according to (4) or (5), further comprising a polyhistidine-containing protein expression vector.
(7) 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.
(8) 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 method for immobilizing a polyhistidine-containing protein comprising specifically binding to a polyhistidine.
(9) 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 method for purifying a polyhistidine-containing protein, comprising specifically binding to a protein and eluting the protein.

Fluorescence image (photograph) showing the binding of His6-GFP to the substrate obtained in each step. (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 | bonding of His6-GFP to the board | substrate obtained in Example 3, 4 and the comparative example 1. 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. 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. Specific examples of 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. Are glass, quartz, carbon, silica gel, graphite, etc., natural polymers are cellulose, cellulose derivatives, alginic acid, alginates, etc., and 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. For example, 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. And it is preferable to couple | bond NTA using the remaining amino group of chitosan. 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).

Hereinafter, a method for producing the NTA-immobilized carrier of the present invention will be exemplified. 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.
In order to wash the glass substrate, it is immersed in 1M sodium hydroxide and treated for 3 days, then washed with ultrapure water three times with acetone and ethanol, and then air-dried. After 3-Aminopropyltriethoxysilan is vapor-deposited on a glass substrate for 2 hours, heat treatment is performed at 100 ° C. for 2 hours. The glass substrate is immersed in a 2.5% glutaraldehyde solution adjusted to pH 7 with a buffer solution HEPES, left to stand overnight, washed with ultrapure water three times and ethanol once and air-dried. The glass substrate is immersed in a 0.05% (w / v) chitosan solution adjusted to pH 8 with HEPES, treated overnight, and then washed with ultrapure water. As a result, 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. Next, 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. As a result, the aldehyde group and the amino group of NTA are bonded, and NTA is fixed on the glass substrate via chitosan.
Note that 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.

By adding a solution of a divalent metal ion to the NTA-immobilized support obtained above, the divalent metal ion can be coordinated with NTA. Here, 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.

  Here, 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. In the present invention, 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. In addition, 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. For example, in the case of an enzyme, it can also be used in an assay for an inhibitor of the enzyme. In the case of an antibody, it can also be used for ELISA or antigen purification. Further, by using 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.

  Further, 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.

  Hereinafter, the present invention will be specifically described with reference to examples. However, this invention is not restrict | limited to the aspect of a following example.

Example 1
An NTA-immobilized substrate was prepared by the following procedure. In addition, the board | substrate after completion | finish of each process was preserve | saved for the fixation efficiency comparison.

Glass substrate cleaning (1M NaOH)
Masked, unmodified glass substrate (MATSUNAMI SLIDE GLASS ring mark) x 6 pieces in 1M NaOH 80ml and left for 3 nights.
↓
Wash 3 times with ultrapure water (Milli-Q water), 1 time with acetone, 1 time with ethanol, and air dry. Store one copy for comparison at the time of GFP (Green fluorescent protein (His6 addition)) spot.
↓
Aminosilane treatment <br/> aminosilane (1% 3-Aminopropyltriethoxysilane) 10ml glass substrate 5 sheets placed under reduced pressure in a desiccator, deposited aminosilane. Leave for 2 hours.
↓
Bake glass substrate at 100 ° C for 2 hours. Save one for comparison at the time of GFP spot.
↓
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.
↓
Clean the glass substrate with ultra pure water. Save one for comparison at the time of GFP spot.
↓
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. 340-08071) to an Eppendorf tube with 0.0015 g and add 15 μl of 1M HEPES (pH8) and 35 μl of ultrapure water to a AB-NTA aqueous solution (300 mM HEPES pH8). Make 50 μl. (Concentration 0.11M)
↓
Take 2 μl from the solution and confirm that the pH is around 8 using a pH test paper.
↓
Spot on a glass substrate at 7μl / well, put parafilm on top and leave in a tapper overnight.
↓
Wash with ultra pure water.
↓
NiCl ₂ treatment A 1.7M NiCl ₂ aqueous solution prepared in advance was spotted at 7 µl / well, and left for 1 hour with parafilm applied from above. As a result, an NTA-immobilized (Ni-NTA) substrate coordinated with Ni ²⁺ ions was obtained.

GFP spot 6 substrates after each step (after NaOH cleaning, after aminosilane treatment, after glutaraldehyde treatment 1, after chitosan treatment, after glutaraldehyde treatment 2 and after AB-NTA treatment) Spot 4 μl of GFP (Green fluorescent protein (His6 added): 3.0 mg / ml) in 1 well, and confirm fluorescence with a transilluminator.
↓
After standing for 1 hour, wash with ultrapure water and check fluorescence. (UV 365nm)
↓
Imidazole elution
Place a Ni-NTA substrate with confirmed fluorescence in 30 ml of 500 mM imidazole and leave it for 3 nights with stirring.
↓
Fluorescence is observed after washing with ultrapure water. Confirm elution of GFP.

The results are shown in FIG.
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.

After coordinating Ni ²⁺ ions to each of the chitosan-NTA modified substrate, glutaraldehyde-NTA modified substrate, and polyallylamine-NTA modified substrate, 4 μl of His6-GFP was spotted, allowed to stand for 1 hour, and then ultrapure water Then, each glass substrate was washed and fluorescence was observed with a transilluminator (ultraviolet light wavelength: 365 nm).

  The results are shown in FIG. As a result, the fluorescence of GFP was almost undetectable by washing with ultrapure water on the glutaraldehyde-NTA modified substrate and polyallylamine-NTA modified substrate, whereas strong GFP was not detected on the chitosan-NTA modified substrate even after washing with ultrapure water. Fluorescence was detected, indicating that His6-GFP was immobilized efficiently.

  The surface area is increased by modifying the glass substrate with chitosan, and the modification efficiency of Ni-NTA is increased because the amino groups present 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/> the glass substrate was placed under reduced pressure with aminosilane (1% 3-Aminopropyltriethoxysilane) in a desiccator, left for two 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.
↓
Chitosan treatment A glass substrate is attached to a chitosan solution (100 mM HEPES pH 8, chitosan (SIGMA Cat. No. 3646, 85% deacetylated) 0.05%) and left at 37 ° C for 1 day with stirring.
↓
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) is spotted on a glass substrate at four locations, left on top in a tapper with parafilm from above.
↓
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/> the glass substrate was placed under reduced pressure with aminosilane (1% 3-Aminopropyltriethoxysilane) in a desiccator, left for two 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.
↓
Chitosan treatment A glass substrate is attached to a chitosan solution (100 mM HEPES pH 8, chitosan (SIGMA Cat. No. 3646, 85% deacetylated) 0.05%) and left at 37 ° C for 1 day with stirring.
↓
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.
↓
Chitosan treatment A glass substrate is attached to a chitosan solution (100 mM HEPES pH 8, chitosan (SIGMA Cat. No. 3646, 85% deacetylated) 0.05%) and left at 37 ° C for 1 day with stirring.
↓
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.

Comparative Example 1
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/> the glass substrate was placed under reduced pressure with aminosilane (1% 3-Aminopropyltriethoxysilane) in a desiccator, left for two 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.
↓
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.
↓
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.
↓
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.
↓
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 over 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.

The fluorescence measurement results before and after washing for Examples 3 and 4 and Comparative Example 1 (n = 4 respectively) are shown in FIG.
Moreover, when a calibration curve was prepared and the amount of GFP immobilized after washing was quantified, it was as shown in Table 1.
As a result, when double-modified with PEIb, GFP was liberated by washing and could hardly be immobilized, but with chitosan modification, GFP was immobilized after washing, indicating that the immobilization efficiency was good. It was. And in the case of chitosan double modification, it was found that the immobilization amount increased more than twice, and immobilization unevenness was reduced.

PEIb二重修飾では固定化量が少なく検量範囲外であった。

With PEIb double modification, the amount of immobilization was small and out of the calibration range.

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.
Further, 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.
For the gold foil substrate, 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.

  According to 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 produce a protein array that is effective for detecting protein interactions. In addition, a large amount of protein can be purified.

Claims (9)

An NTA-immobilized carrier comprising chitosan immobilized on a carrier and nitrilotriacetic acid (NTA) (chitosan-NTA) bound to the chitosan, containing a divalent metal ion and containing polyhistidine An NTA-immobilized carrier that can bind proteins. The 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. NTA immobilization support. The NTA-immobilized carrier according to claim 1 or 2, wherein the carrier is a substrate, and chitosan-NTA is immobilized at a plurality of locations on the substrate. A kit for purifying or immobilizing a polyhistidine-containing protein comprising the NTA-immobilized carrier according to any one of claims 1 to 3. The kit according to claim 4, further comprising a divalent metal ion solution. The kit according to claim 4 or 5, further comprising a polyhistidine-containing protein expression vector. A protein chip comprising the NTA-immobilized carrier of claim 3 and having a polyhistidine-containing protein bound to a plurality of locations on the NTA-immobilized carrier. A bivalent metal ion is coordinated to the NTA-immobilized carrier according to any one of claims 1 to 3, and then a polyhistidine-containing protein-containing sample is loaded on the carrier, so that the polyhistidine-containing protein is loaded onto the carrier. A method for immobilizing a polyhistidine-containing protein, comprising specifically binding. A bivalent metal ion is coordinated to the NTA-immobilized carrier according to any one of claims 1 to 3, and then a polyhistidine-containing protein-containing sample is loaded on the carrier, so that the polyhistidine-containing protein is loaded onto the carrier. A method for purifying a polyhistidine-containing protein, comprising specifically binding and eluting the protein.

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