Background Art Metal chelate composition, which is represented by ethylenediamine, ETDA (ethylenediamintetraacetin acid), dimercaptol (2, 3-dimercapto-1-propanol), etc. , means the composition combined with metallic ion of 2 valencies in oxidation state.

Metal chelate composition is used in various ways in the elimination of heavy metal, the treatment of wastes and metal affinity chromatography and so on.

Technology of metal affinity chromatography as a kind of affinity chromatography (Porath, J. 1975, Nature 258: 598-599) is a technology of purification metal affinity material using matrix of chelate ligand complexed or macromolecule. Since the histidine of imidazole among amino acids is linked by a coordinate covalent bond with metallic ion, its characteristics is used in the establishment of the technology of the purification of the protein having histidine.

Various kind of metal chelate composition is used in protein purification chromatography. Metal chelate composition of ligand such as imminodiacetic acid, nitrilotriacetic acid (NTA), carboxymethyl aspartic acid, etc. linked with matrix of macromolecule is produced commercially.

However, conventional metal chelate compositions currently used in affinity chromatography have their own merits and demerits.

Since, in imminodiacetic acid composition, metal and ligand are linked by three coordinate covalent bonds, it is necessary to pay attention in use because it is easily detached by hydrogenation etc. (Porah, J. 1983, Biochemistry 22: 1621-1630).

Carboxymethyl aspartic acid composition has the demerit of difficulty in absorption of a sample to resin due to weak combination force between metal attached to legand and protein (Chaga, G. 1999, Biothechnol. Appl. Biochem. 29: 19-24).

Nitrilotriacetic acid (NTA) is a legand that has the strongest legand affinity among the chelate compositions currently used and transition metal combination. Patent (U. S.

4, 877, 480) by Hoffman la Loche brings out the following: the composition of NTA is a combination of 3 covalent bonds of carboxymethyl radicals to nitrogen atom. The following composition is adopted in U. S. patent 4, 877, 830 in order to combine such composition of NTA to matrix.

Formula 1 NH2- (CH2) 4-CH (COOH) -N (CH2COOH) 2 + matrix= matrix-linker-NH- (CH2) 4-CH (COOH)-N (CH2COOH) 2 In order to make ligand matrix with the above composition, ligand compound is produced and then combined to the matrix attached with activated linker. Ligand is produced through the hydrogenation after producing N-carboximethylated Cbz-Lysine by the reaction of Cbz-Lysine and bromoacetic acid.

However, the processes of many times of crystallization and recrystallization, drying and dangerous hydrogenation are necessary. And the demerit of it is the excessive cost for producing ligand.

Disclosure of the Invention Therefore, the purpose of the present invention is to overcome the demerit of the conventional metal chelate compositions by deriving the macromolecule complex, wherein nitrilotriacetic acid (NTA) ligand is combined with macromolecule compound in a novel way; ie, to provide the macromolecule complex of metal chelate compositions which can be produced at a low cost by a simple process.

And the purpose of the present invention is to provide the macromolecule complex of matrix type metal chelate composition for chromatography which enables to effectively refine protein by the more rigid combination of metal and chelate composition.

Moreover, it is to provide metal chelate composition having strong affinity to the molecule to make affinity with ligand and macromolecule complex.

Furthermore, it is to provide a researcher with metal chelate composition which can be used by attaching various type of desired matrix and macromolecule to the linker.

In order to achieve the above the above purpose, the inventor derived a process for manufacturing chelate ligand having nitrilotriacetic acid (NTA) composition and-SH as a reaction radical by using amino acid having-SH (sulfhydril) such as Cystein; and suggest

a process for manufacturing macromolecule complex of a novel NTA metal chelate composition.

In the novel metal chelate composition of the present invention, 3 carboxyl radicals of the ligand part are linked to metal and a sulfhydril is linked to other composition, especially macromolecule matrix.

The present invention relates to: the processes of manufacturing the macromolecule complex of metal chelate composition by manufacturing the novel metal chelate composition and linking it to macromolecule; and the macromolecule complex of the metal chelate composition by compounding ligand to macromolecule in regular sequence: and the method of using the above novel metal chelate composition or the macromolecule complex in purification material such as protein.

The terms such as ligand, matrix, macromolecule, metal chelate composition, and macromolecule complex of metal chelate composition are used in"the summary of the invention"and"what is claimed is". The"macromolecule"in the present description means to be the macromolecule before the direct linking of the affinity ligand part to the material (e. g. protein) to be chemically attracted. For example, protein, fat, carbohydrate, alkaloid, resin or cross-linked agarose, cross-linked cellulous, etc. are called macromolecules. That is, the"macromolecule"in the present description is used in a broader meaning which includes general term"matrix"or"resin"as used in the conventional affinity chromatography.

Terms"ligand"and"metal chelate composition"are used together in the present description; also, mean the materials showing affinity to the specific material such as nitrilotriacetic acid.

"Macromolecule complex of metal chelate composition"is used as a term expressing the state of linking of the linker and the metal chelate composition to macromolecule ; also, means the state of transition metal chelated or the state of transition metal excluded.

The process of manufacturing macromolecule of the metal chelate composition of the present invention is a method to attach metal chelate composition manufactured such as N, N dicarboximethyl cystein to macromolecule (Embodiment 1-3).

Another way of manufacturing macromolecule complex of the metal chelate composition is to attach amino acid having-SH such as cystein; carboxcilate amin radical attached, and nitrilotriacetic acid ligand in order to chemically stabilized macromolecule.

(Embodiment 4-7).

The control of pH is important in order to produce desired macromolecule in such a sequential reaction. In the reaction of amino acid with-SH to be linked to macromolecule, acidity should be maintained between Ph 6. 0-8. 5 so that only the-SH enters into reaction and the amin radical does not. And in the carboxilation reaction of

amin acidity should be maintained for proper reaction.

The present invention relates to the metal chelate composition as shown in the following chemical formula. The the metal chelate composition provides researcher with necessary use by linking various type of desired macromolecule and matrix to various linker.

Formula 1 HS-Ri-CH (COOH)-N- (CH2COOH) 2 (wherein, Rl is an alkyl radical linker having 1-5 carbon such as-CH2-or-CH2CH2-.) The present invention relates to metal chelate composition linked with transition metal ion such as Co, Ni, Zn, Fe, Cu, etc. to the above metal chelate composition.

It also relates to the purification process for protein having affinity with metal such as recombinant protein having histidine marker utilizing macromolecule complex of the above metal chelate composition produced. Moreover, it is quite natural that a person having knowledge in the field of the present invention can refine various metal affinity material or selectively make affinity absorption using macromolecule complex of the metal chelate composition of the present invention.

Furthermore, it relates to the macromolecule of the metal chelate composition as shown in the following formula.

Formula 2 R3-R2-S-Rl-CH (COOH) N (CH2COOH) 2 In the above formula, Ri is an alkyl radical linker having 1-5 carbons as in-CH2-or- CH2CH2- ; R2 is a linker activating macromolecule and linking with-SH, preferably as- CH2CH (OH) CH2-,-CH (OH) CH2-0- (CH2) 4CH (OH) CH2-,-CH2CH2S02CH2CH2-,- COCH2-,-CH2CH2CH2-,-CH2CH (OH) CH2-,-CH2CH (Br) CH2-or-OCO-, R3 > is a macromolecule compound including matrix for chromatography4 such as cross-linked agarose, cross-linked cellulose, cross-linked dextran, cross-linked metacrilamide, silica or protein, fat, carbohydrate, alkaloid, resin.

Furthermore, the present invention relates to macromolecule complex of metal chelate composition linking transition metal ion such as Co, Ni, Zn, Fe, Cu etc. with macromolecule complex of the above metal chelate composition as shown in the following chemical formula 3.

Formula 3

M stands for transition metal on the above formula.

The present invention relates to the process of manufacturing macromolecule complex of the above metal chelate composition according to the following sequence.

1) To activate macromolecule using epichlorohydrin 2) To wash the macromolecule with distilled water, buffer solution 3) To have the above macromolecule and amino acid having-SH, preferably cystein or homocystein react. To maintain the reaction for 24 hours preferably in a tightly sealed container in the normal temperature after adding nitrogene.

4) To wash the macromolecule linked with the above-SH, more preferably in a sequence of distilled water, acetic acid, NaHCO3 solution.

5) To carboxilate by bromoacetic acid, etc. Preferably to leave as it is for approx. 12 hours in the normal temperature after mixing with NaOH (pH 10-11), 1M NaHC03 (pH 10.0) in addition to bromoacetic acid.

6) To wash.

The method of manufacturing macromolecule complex of the above metal chelate composition of the present invention is remarkably simpler process than described in the process of the conventional U. S. Patent, and provides macromolecule complex of the metal chelate composition having similar absorption effect of to conventional NTA matrix with less time and lower cost.

The present invention describes a manufacturing method of linking metal chelate composition to activated macromolecule after the metal chelate composition is composed; and illustrate that the 2 novel methods can be used in the same manner.

Brief Description of Drawings Fig. 1 shows a electrophoresis photograph of the purification process of the MutS protein attached to the end of 6 histidine by using metal chelate agarose of the present invention and NTA agarose.

Fig. 2 shows a electrophoresis photograph of the purification process of the same protein by using the above 2 metal chelate agarose again.

M: size marker of NOVEX corp.

1-4 : purification process by using metal chelate resin of the present invention.

5 ~ 8 : purification process by using NTA agarose resin of QIAGEN Corp.

1,5 : loaded sample 2,6 : samples left unabsorbed and floen out.

3,7 : samples after being washed by 50mM imidazole 4,8 : samples eluted by 200mM imidazole Best Mode for Carrying Out the Invention The composition of the present invention is described through the embodiments as follows. However, the scope of the invention is not limited to the descriptions of the following embodiments Example 1 The present embodiment is an example of the reaction producing metal chelate composition.

10mg of S-trityl-cysteine is melt in IN NaOH. 16g of bromoacetic acid is added gradually to IN NaOH. After being mixed for 2 hours it is agitated for 12 hours in the normal temperature. Subsidence is made by adding IN HCL after the sediments being gathered with glass filter funnel and dissolved in 20mtof 1N NaOH (N, N- dicarboximethyl-cysteine).

The reaction is maintained for 1 hour after putting the subsidence into a triangle flask, adding 100g of distilled water and lml of trifluoride acetic acid, and filling nitrogen in it.

The subsidence is recovered by adding acetone of three times of its volume. The subsidence is lyophilized after being washed. Thus, N, N-dicarboximethyl- cysteine can be produced.

Example 2 The embodiment is another example of the reaction producing metal chelate composition. lOg of cystine or homocystine is dissolved in the 50ml of the solution of 0. 1M NaHCO3, pH 10.5. After dissolving 24g of bromoacetic acid into 40ml of 4N NaOH solution and adjusting pH to 10.5. It is added to cystine or homocystine solution.

Example 3 The present embodiment is another example of the reaction linking N, N. dicarboxymethyl-cysteine produced in the above embodiment 1 or 2 to macromolecule lOg of activated macromolecule produced as in the embodiments 4,5, 6 is dissolved in 100ml of 25mM NaPi (pH 7.5). After 100mg of N, N. dicarboxymethyl-cysteine is dissolved in 100ml of 25mM NaPi (pH 7.5), it is mixed with the above activated macromolecule solution. After being mixed for 24 hours in the natural temperature, the macromolecule is separated by glass funnel and washed with 1000ml of distilled water, 1000ml of 1% acetic acid, 1000ml of distilled water in order.

Example 4 The present embodiment is an example of the reaction linking R2 by activation of ceparose CL-6B, a cross-linked agarose as macromolecule.

100mg of ceparose CL-6B matrix is washed using glass funnel by 1000ml of distilled water. Macromolecule matrix in which water is eliminated is moved into the 500ml triangle flask, and is added by 40ml of 4N NaOH and 15ml of epichlorohydrin. It is agitated for 6 hours in the temperature of 28 °C after plugging the opening with a silicon plug. The reacted solution is move into glass filter funnel and washed by 1000mg of distilled water.

Example 5 The present embodiment is an example of the reaction linking the other linker R2 100ml of ceparose CL-6B matrix is washed by 1000ml of distilled water using glass funnel. Macromolecule matrix in which water is eliminated is moved into the 500ml triangle flask, and is added by 0.6M NaOH containig 2mg/m of NaBH4 and. 4m. of BDE (butanedioldiglycidyl ether, 60% purity). It is agitated for 8 hours at the

temperature of 28 °C and is moved to the glass funnel. The reacted solution is move into glass filter funnel and washed by 1000mg of distilled water.

Example 6 The present embodiment is an example of the reaction linking the other linker R2 1.4g of bromoacetic acid is put into triangle flask together with 1.4g of N- hydroxysuxineimide 80mQ of dioxane and agitated. It is agitated for 70 minutes after adding 2.2g of dicyclohexylcarbodiimide.

Sediment is eliminated by glass funnel. Bromoreceticliydroxysuxineimide is composed by eliminating dioxane with a rotary evaporator. 3g of AH-ceparose is washed by 100mQ of distilled water in the glass funnel filled with 0.1 sodium phosphate solution (Ph 7.5) ; and agitated for 2 hours after adding 2mg of the bromorecetichydroxysuxineimide prepared as in the above. The solution is moved into glass funnel and washed with 100mQ of distilled water.

Example 7 The present embodiment is an example of the reaction linking metal chelate composition with the activated macromolecule produced in the above embodiments 4,5, 6.

100mQ of the macromolecule activated as in the process of the embodiments 4,5, 6 is put into triangle flask. 100mQ of 25mM sodium phosphate pH 7.5 solution. After dissolving lOg of L-cystein or L-homocystein into 100mQ of 25mM sodium phosphate solution, pH of it ia adjusted to 7.5. It is agitated for 24 hours after being filled with nitrogen gas in the reactor. The solution is moved into glass filter funnel and is washed with 100mQ of distilled water, 1000mQ of 10% acetic acid, distilled water in sequence.

The washed macromolecule moved into triangle flask is added with 100mQ 1M NaHCO3, pH 10.0 ; and the solution of 100mQ of 4N NaOH where 15 g of bromoacetic acid is dissolved and pH adjusted to 10.5. The solution is agitated for 24 hours in the normal temperature, moved into funnel and washed with 1000mg of distilled water, 1000mu of 10% acetic acid, distilled water in sequence.

Example 8 The present embodiment is described for the method for attaching transition metal to the macromolecule attached with metal chelate composition and the method for

measuring density of metal ion.

100ml of the macromolecule complex of the metal chelate composition prepared in the embodiment 3 or 7 is put into glass filter funnel and is washed with 500ml of distilled water. After 100ml of the solution 100mM EDTA, pH 8.0 is added, the solution liquated is recovered. It is found that there exist nickel ion of more than 560, ug/ml in the result of measurement by an atomic absorption spectrophotometer.

Example 9 The present embodiment is an example of the purification of protein by using chelate agarose which is a macromolecule complex of the metal chelate composition of the present invention as prepared in the above embodiment 8.

0. 5ml of the chelate agarose of the present invention is put into chromatography tube and washed with 5ml of distilled water. It is washed with 5ml of the solution of 5 mM imidazole, 0.5 M NaCl, 50mM NaH2PO4, pH 8. 0 after being washed with 5ml of 100mM NiS04 solution After MutS protein where 6 histidine are attached at end of amino is manifested in 1000ml of colon bacilli, cells are recovered by centri-fugation.

Cells are put into 50ml of crush buffer solution (0. 15M NaCl, 50mM NaH2PO4, pH 8.0) and crushed by ultrasonic crusher. The solution is centrifuged for 10 minutes by 10,000g ; 10ml of higher layer liquid is poured into chelate agarose chelated with nickel.

After protein which is not attached to metal chelate agarose being eliminated by 5ml of the solution of 5mM imidazole, 0. 5M NaCl, 50mM NaH2PO4, pH 8. 0, contaminated protein attached weakly is eliminated by 50ml of the solution of 50mM imidazole, 0. 5M NaCl, 50mM NaH2PO4, pH 8. 0. histidine marker protein which is attached to the metal chelate agarose is separated by 5ml of the solution of 200mM imidazole, 0. 5M NaCl, 50mM NaH2PO4, pH 8.

The purity is confirmed by Coomasie blue after the specimen of each step is separated by 12% SDS-PAGE (Fig. 1). The amount of the protein attached to the metal chelate agarose is measured using the Bradford method with the standard of small serum albumin.

Collected fraction is diluted 100 times with distilled water and light absorbing index is measured at 600nm on the 0. lml of the liquid and compared with the normal protein.

MutS protein is attached on the macromolecule of the novel metal chelate composition in the ratio of 5. 2mg per lmQ. The NTA agarose, macromolecule complex of the metal chelate composition of QIAGEN corp. is absorbed by 5. lmg.

The metal chelate agarose used in the purification is set to equilibrium by using 5mM imidazole, 0. 5M NaCl, 50mM NaH2PO4, pH 8. 0 solution 5mQ after washing with 200mM of imidazole, 0. 5M NaCl, 50mM NaH2P04, pH 8.0 solution 10mu. Cell crushing solution 2mg is added to the matrix and the matrix is washed with 5mM imidazole, 0. 5M NaCl, 50mM NaH2PO4, pH 8.0 solution 5ml. By washing and separating with the same method as above the specimen is confirmed by electrophoresis (Fig. 2). Fig. 2 shows the result after reusing the metal chelate agarose and commercial metal chelate NTA agarose of QIAGEN corp. , which is similiar to the result of Fig. 1. This shows that the metal chelate composition of the present invention is very stable and can be used repeatedly.

Industrial Applicability As described above, the metal chelate composition, the macromolecular complex and the method for the composition are carried out very simply compared with the method of prior art, reducing time and cost while showing similar properties of prior art metal chelate resins.