KR100401339B1 - Metal chelate ligand - Google Patents

Abstract

PURPOSE: Provided is a metal chelate resin, which can be obtained by a simple process at a low cost and is capable of forming a strong bond between a metal and a ligand so as to purify proteins efficiently. CONSTITUTION: The metal chelate ligand is based on the bonding of nitrilotriaceticacid(NTA) ligand with a polymer matrix. The metal chelate ligand uses the difference between chemical reactivity of a -SH group and that of an amine group in a peptide having a glutathione sulfurhydril(SH) group. The metal chelate ligand is represented by the following formula: (CH2COOH)2-N-CH(COOH)-CH2CH2-CO-NH-CH(CH2SH)-CO-NH-CH2(COOH).

Description

Metal chelate ligand

The present invention relates to a novel metal chelate resin, a method for preparing the same, and a method for using the novel metal chelate resin in the purification of substances such as proteins, using glutathione (γ-glutamyl-cysteinyl-glycine) as a base material. By using a metal chelate ligand represented by the formula (CH ₂ COOH) ₂ -N-CH (COOH) -CH ₂ CH ₂ -CO-NH-CH (CH ₂ SH) -CO-NH-CH ₂ (COOH) It relates to a novel metal chelate resin produced.

Metal affinity chromatography technology is a kind of affinity chromatography (Porath, J. 1975, Nature 258: 598-599), which uses a resin attached to a chelate ligand to purify metal-affinity materials. Since the imidazole group of histidine in amino acids coordinates with metal ions, a technique for purifying proteins having histidine using this property has been established.

Several metal chelate ligands are used for protein purification chromatography. Resin using a ligand such as iminodiacetic acid (niminodiacetic acid), nitrilotriacetic acid (NTA), carboxymethyl aspartic acid is commercially produced.

However, there are advantages and disadvantages to the metal affinity chromatography resins currently used (combined herein with the same meaning as "metal chelate resins"). Iminodiacetic acid resins should be used with caution because they are easily dissociated by hydration and the like because metal and ligand are linked by three coordination bonds (Porah, J. 1983, Biochemistry 22: 1621-1630).

In addition, carboxymethyl aspartic acid resin has a weak binding strength between the metal and the protein attached to the ligand, so it is not easy to adsorb the sample to the resin (Chaga, G. 1999, Biothechnol.Appl. Biochem. 29: 19- 24).

Nitrilotriacetic acid ligands have strong metal-ligand, metal-protein bonds. However, there are disadvantages in that the raw material of the modified ligand, which is required to attach the ligand to the resin, is expensive and additional time and equipment are required (USP4,877,830).

Sigma-Aldrich's WO01 / 81365, which has a nitrilotriacetic acid (NTA) and simplifies the manufacturing process using the amino acid cysteine, is a compound having a metal chelate group that is short when combined with a protein. It is predicted that purification efficiency is low because of the high possibility of steric hinderance.

Nitrilotriacetic acid (NTA) is a ligand having the strongest ligand affinity and transition metal bond among the metal chelate resins currently in use. US Patent No. 4,877,830 to Hoffman la Loche discloses the following. The structure of NTA is a covalent bond of three carboxymethyl groups to a nitrogen atom, and the following structure was chosen to bind NTA of this structure to a matrix.

Matrix-Linker-NH- (CH ₂ ) ₄ -CH (COOH) -N (CH ₂ COOH) ₂

In order to make the resin having the above structure, a ligand was first made, and then a ligand was attached to the resin to which the activated linker was attached. To do this, the following 11 steps are required.

1. Dissolve 42 g of Cbz-Lysine in 225 ml 2N NaOH and cool.

2. Dissolve 41.7 g bromo acetic acid in 150 ml of 2N NaOH and slowly add to solution # 1.

3. After 2 hours, transfer the solution to room temperature and overnight.

4. After heating at 50 ° C. for 2 hours, add 540 ml of 1N NaOH.

5. Recover the precipitate after cooling.

6. The precipitate is dissolved in 1N NaOH and precipitated again by adding 1N HCl.

7. Recover the precipitate and dry it.

8. Dissolve 7.9 g of the precipitate in 49 ml of 1N NaOH solution, add a small amount of 5% Pd / C, and then hydrogenate at room temperature and normal pressure. The precipitate is removed from the reaction and then dried.

9. 100 ml Sepharose CL-6B resin activated with Epibromohydrin, 6.5 g N- [5 amino-1-carboxypentyl] -iminodiacetic acid (hydrogenation product), 50 ml DW. After adding 10.6 g sodium carbonate, the mixture is slowly stirred overnight at 60 ° C.

10. Wash resin in order of 500 ml of distilled water, 100 ml of 2% NiSO ₄ , 200 ml of distilled water, 200 ml of 0.2 M acetic acid (containing 0.2 M NaCl and 0.1% Tween 20) and 200 ml of distilled water.

11. Obtain a resin filled with Ni ions at 7.1 µM / ml.

However, the hydrogenation process is dangerous and complex. And after drying, the process of recrystallization using ethanol is required.

Roche's U.S. patent adopts N-linked NTA as a metal affinity resin, while U.S. Pat.No. 5,932,102 is a resin marketed under the TALON trademark, which binds an amino group of aspartic acid to an activation matrix and then carboxymethylates.

COOH

I

Matrix-Linker-N-CH-CH ₂ COOH

I

CH ₂ COOH

This resin is structurally similar to Roche's NTA, but has a problem in that the binding force between the metal and the adsorbent is very weak.

Accordingly, the present invention aims to overcome the drawbacks of conventional metal chelate resins by designing new modified ligands that bind the nitrilotriacetic acid (NTA) ligand with the polymer matrix. That is, the present invention is to provide a metal chelate resin that can be manufactured at a low cost in a simple process.

In addition, the present invention is to provide a metal chelate resin that can be purified more effectively by binding the metal and ligand more tightly.

In addition, it is an object of the present invention to provide a metal chelate resin having a strong adsorptive force between a molecule to be adsorbed and a resin.

In addition, the present invention seeks to provide an adsorbent ligand that can be used by attaching to various types of matrices and linkers desired by the researcher.

FIG. 1 is an electrophoretic photograph of a process of purifying MutS protein having 6 histidines attached to its terminal using NTA agarose resin of metal chelate resin and Qiagen of the present invention.

M: Novex (NOVEX) size markers,

1: resin input fraction,

2-5: NTA-agarose resin of the present invention, 6-9: existing NTA-agarose resin,

2, 6: nonadsorbed fraction,

3, 7: washing fraction,

4, 8: 50 mM imidazole fraction,

5, 9: 200 mM imidazole fraction

In order to achieve the above object, the present inventors focused on obtaining a new NTA metal chelate resin by using a difference in chemical reactivity between the -SH group and the amine group of a peptide having a glutathione -SH (sulfurhydril) group.

The present invention relates to novel metal chelate resins, methods for their preparation and methods for using the novel metal chelate resins in the purification of substances such as proteins.

In the description and in the claims, the terms resin, ligand and matrix are used. As used herein, the term "matrix" refers to a polymer matrix prior to the binding of an affinity ligand moiety that directly binds to a substance (such as a protein) to be adsorbed. For example, crosslinked agarose, crosslinked cellulose and the like are called matrices.

In addition, the term "resin" is used herein to express a state in which a linker and an affinity ligand moiety are bound to a matrix.

In addition, in the present specification, "ligand" refers to a material that can exhibit adsorptivity to a specific material, such as nitrilotriacetic acid.

In addition, the term "metal chelate resin" as used herein refers to a resin capable of chelating a transition metal, and means a state in which the transition metal is chelated or does not include a transition metal.

The method for preparing the metal chelate resin of the present invention sequentially attaches a peptide having a glutathione-SH group to a chemically activated polymer matrix and then carboxymethylates the amine group of the attached peptide to nitrilo triacetic acid ligand. (Examples 1-4).

Another method for preparing the metal chelate resin of the present invention is to prepare a chelating ligand such as N, N dicarboxymethyl-glutacyion and then attach it to the polymer matrix (Examples 5-7).

In this sequential reaction, control of pH is important to make the desired compound. In the reaction in which the peptide having the -SH group binds to the polymer matrix, the acidity must be maintained at pH 6.0-8.5 so that only the -SH group participates in the binding reaction and the amine group does not participate in the reaction. In addition, an appropriate reaction occurs only when the carboxymethylation of the amine group is maintained at pH 9-12.

In addition, the present invention relates to a metal chelate ligand represented by the following formula. By using this metal chelate ligand, it can be attached to various kinds of matrices and linkers desired by the researcher and used for necessary purposes.

(CH ₂ COOH) ₂ -N-CH (COOH) -CH ₂ CH ₂ -CO-NH-CH (CH ₂ SH) -CO-NH-CH ₂ (COOH)

The present invention also relates to a method for purifying a protein having affinity with a metal, such as a recombinant protein having a histidine label, by chromatography using a metal chelate resin prepared as described above. Of course, in addition to purifying or selectively adsorbing various metal affinity materials using the metal chelate resin of the present invention will be apparent to those skilled in the art.

The present invention relates to a metal chelate resin represented by the following formula.

(CH ₂ COOH) ₂ -N-CH (COOH) -CH ₂ CH ₂ -CO-NH-CH ₂ (CH ₂ SR ₁ -R ₂ ) -CO-NH-CH ₂ (COOH)

In the above formula, R ₁ is a linker that activates the polymer matrix and binds to -SH group, and preferably -CH ₂ CH (OH) CH _2- , -CH (OH) CH ₂ -O- (CH ₂ ) ₄ CH ( OH) CH _2- , -CH ₂ CH ₂ SO ₂ CH ₂ CH _2- , -COCH _2- , -CH ₂ CH ₂ CH _2- , -CH ₂ CH (Br) CH ₂ -or -OCO-, and R ₂ is a polymer matrix including crosslinked agarose, crosslinked cellulose, crosslinked dextran, crosslinked methacrylamide, a matrix for chromatography such as silica or glass, and the like.

The present invention also relates to a metal chelate resin in which transition metal ions such as Co, Ni, Zn, Fe, Cu, etc. are bonded to the metal chelate resin.

Provided that M represents a transition metal.

The present invention also relates to a method for preparing the metal chelate resin according to the following sequential steps.

1) The polymer matrix is activated using epichlorohydrin.

2) After that, wash the matrix with distilled water, buffer solution and the like.

3) Glutathione having the -SH group is reacted with the matrix. The reaction is preferably carried out at room temperature for 24 hours after adding nitrogen to a sealed container.

4) Wash the matrix bound to the glutathione having the -SH group. Preferably, distilled water, acetic acid 10%, distilled water, NaHCO ₃ solution in the order of washing.

5) Carboxymethylation with bromo acetic acid or the like. Preferably mixed with NaOH (pH 10-11), 1M NaHCO ₃ (pH 10.0) in addition to bromo acetic acid and then overnight at room temperature.

6) Wash.

The metal chelate resin manufacturing method of the present invention can provide a metal chelate resin exhibiting a similar adsorption effect similar to that of the conventional NTA resin, while the process is significantly simpler and time and cost is reduced compared to the resin preparation method disclosed in the prior US patent. have.

In addition, the present invention describes a method for preparing a ligand to be synthesized first and then bound to an activated matrix, illustrating that both new methods can be used in the same way.

Hereinafter, the configuration of the present invention in detail through the embodiment. However, the scope of the present invention is not limited only to the description of the following examples.

<Example 1>

This example is a reaction example in which R ₁ is bound by activating Sepharose CL-6B, which is crosslinked agarose as a polymer matrix.

100 ml of Sepharose CL-6B matrix was washed with 1000 ml of distilled water using a glass funnel. The water-free matrix was transferred to a 500 ml Erlenmeyer flask, and 40 ml of 4N NaOH and 15 ml of epichlorohydrin were added thereto. The inlet was closed with a silicone stopper and stirred at 28 ° C. for 6 hours. The reaction solution was transferred to a glass filter funnel and washed with 1000 ml of distilled water.

<Example 2>

In another embodiment it is a reaction example to link another linker R ₁ .

100 ml of Sepharose CL-6B matrix was washed with 1000 ml of distilled water using a glass funnel. The water-free matrix was transferred to a 500 ml Erlenmeyer flask, and 2 mg / ml of NaBH ₄ and 0.4 ml of BDE (butanedioldiglycidyl ether, 60% purity) in 0.6 M NaOH were added. After stirring at 28 ° C. for 8 hours, the reaction solution was transferred to a glass filter funnel and washed with 1000 ml of distilled water.

<Example 3>

In another embodiment it is a reaction example to bond another linker R ₁ .

1.4 g of bromo acetic acid was put into a Erlenmeyer flask with 1.4 g of N-hydroxysuccinimide and 80 ml of dioxane, followed by stirring. 2.2 g of dicyclohexylcarbodiimide was added, followed by stirring for 70 minutes. The precipitate was removed with a glass funnel. The dioxane was removed with a rotary evaporator to synthesize bromoacetic hydroxysuccinimide. 3 g of AH-Sepharose was washed with 100 ml of distilled water in a glass funnel, and then transferred to an Erlenmeyer flask containing 0.1 M sodium phosphate solution (pH 7.5), and 2 g of bromoacetic hydroxysuccinimide prepared above was added thereto, followed by stirring for 2 hours. It was. The solution was transferred to a glass funnel and washed with 100 ml of distilled water.

<Example 4>

Example 4 is a reaction example in which a chelate ligand is bound to the activated polymer matrix prepared in Examples 1, 2, and 3.

100 ml of the activated matrix was added to the Erlenmeyer flask in the same manner as in Example 1, 2 or 3. 100 ml of 25 mM sodium phosphate pH 7.5 solution was added. 10 g of glutathione was dissolved in 100 ml of 25 mM sodium phosphate solution, and the pH was adjusted to 7.5. Nitrogen gas was charged into the reactor and stirred for 24 hours. The solution was transferred to a glass filter funnel and washed with distilled water 100 mL, 10% acetic acid 1000 mL and distilled water in that order. The washed resin was transferred to an Erlenmeyer flask, 100ml 1M NaHCO ₃ , pH 10.0 was added, 15g of bromo acetic acid was dissolved in 100ml of 4N NaOH solution, and the solution was adjusted to pH 10.5. Stir at room temperature for 24 hours. The solution was transferred to a glass funnel and washed in the order of 1000 ml of distilled water, 1000 ml of 10% acetic acid and distilled water.

Example 5

This example is a reaction example in which the chelating ligand is not bound to the matrix.

10 g of S-trityl-glutathione was dissolved in 1N NaOH. A solution of 16 g bromo acetic acid dissolved in 1N NaOH was slowly added to the cooled solution. After stirring for 2 hours, the mixture was stirred at room temperature for 12 hours. 1N HCl solution was added slowly to precipitate. The precipitate was collected with a glass filter funnel and dissolved again in 20 mL of 1N NaOH and added with 1N HCl to form a precipitate (N, N-dicarboxymethyl-S-trityl-glutacyion). The precipitate was placed in an Erlenmeyer flask, and 100 ml of distilled water was added, 1 ml of trifluoroacetic acid was added, and the mixture was reacted for 1 hour after filling with nitrogen. Three times the volume of acetone was added and the precipitate was recovered. The precipitate was washed with acetone and then lyophilized. Thus, N, N dicarboxymethyl-glutacyion was obtained.

<Example 7>

As another example, N, N dicarboxymethyl-glutathione obtained in Example 5 is a reaction example in which the polymer matrix is bonded.

10 g of the activated polymer matrix prepared in Examples 1, 2 or 3 was dissolved in 25 mM NaPi, 100 ml (pH 7.5). 100 mg of N, N dicarboxymethyl-glutacyion was dissolved in 100 ml of 25 mM NaPi (pH 7.5) and mixed with the activated polymer matrix solution. After stirring at room temperature for 24 hours, the resin was separated with a glass funnel and washed in the order of 1000 ml of distilled water, 1000 ml of 1% acetic acid and 1000 ml of distilled water.

<Example 8>

This embodiment is an example of attaching a transition metal to a resin to which a ligand is attached and measuring the metal ion concentration.

100 ml of the metal chelate resin obtained in Example 4 or 7 was added to a glass filter funnel and washed with 500 ml of distilled water. After washing with 200ml of 100mM NiSO ₄ solution was washed with 500ml of distilled water. After adding 100 ml of 100 mM EDTA, pH 8.0 solution, the eluted solution was recovered.

As a result of quantification using an atomic absorption spectrometer, it was confirmed that there were 560 µg / ml or more of nickel ions.

Example 9

This example is an example of protein purification using the metal chelate resin of the present invention prepared in Example 4 or 7.

0.5 ml of the metal chelate resin of the present invention was placed in a chromatography tube and washed with 5 ml of distilled water. 5 ml of 100 mM NiSO ₄ solution followed by 5 ml of 5 mM imidazole, 0.5 M NaCl, 50 mM NaH ₂ PO ₄ , pH 8.0 solution.

MutS protein with six histidines at the amino terminus was expressed in E. coli 1000ml, and the cells were recovered by centrifugation. The cells were placed in 50 ml of lysis buffer (0.15 M NaCl, 50 mM NaH ₂ PO ₄ , pH 8.0) and disrupted with an ultrasonic crusher. The solution was centrifuged at 10,000 g for 10 minutes, and then 10 ml of the supernatant was injected into nickel chelated metal chelate resin. 5 mM imidazole, 0.5M NaCl, 50 mM NaH ₂ PO ₄ , pH 8.0 solution to remove unattached protein and contaminated protein weakly adhered to 5 ml of 50 mM imidazole, 0.5M NaCl, 50 mM NaH ₂ PO ₄ , pH 8.0 solution Was removed. Histidine-labeled protein attached to the resin was isolated with 5 ml of 200 mM imidazole, 0.5 M NaCl, 50 mM NaH ₂ PO ₄ , pH 8.0 solution.

Samples for each step were separated by 12% SDS-PAGE and confirmed purity by Coomassie blue staining (FIG. 1). The amount of protein attached to the resin was quantified by bovine serum albumin using the Bradford method. The recovered fraction was diluted 100-fold with distilled water, 0.1 ml was taken, and the same amount of Bradford reagent was added. After 5 minutes, the absorbance was measured at 600 nm and compared with the standard protein.

The novel metal chelate resin of the present invention adsorbed 5.2 mg of MutS protein per ml. QIAGEN resin was adsorbed at 5.1 mg.

Accordingly, the present invention can provide a metal chelate resin and a method for producing the same, which is significantly simpler than the resin manufacturing method disclosed in the prior US patent, and exhibits an adsorption effect similar to that of the conventional NTA resin while reducing time and cost. .

Claims (1)

Metal chelate ligand represented by the following formula. (CH ₂ COOH) ₂ -N-CH (COOH) -CH ₂ CH ₂ -CO-NH-CH (CH ₂ SH) -CO-NH-CH ₂ (COOH)