JPH0797988B2 - Method of immobilizing substances - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for immobilizing a substance. Specifically, a method for immobilizing a substance, which comprises cross-linking a substance and a carrier with a polyvalent crosslinking compound having two or more maleimide groups in one molecule or both a maleimide group and a succinimide ester group in one molecule. Is.

According to the present invention, various substances such as low-molecular substances, high-molecular substances, physiologically active substances, proteins, enzymes, ligands, antigens, antibodies, microbial cells, and cells can be freely immobilized on a carrier. The present invention can be widely used in the technical fields of purification, production, biosynthesis, analysis and measurement.

For example, when the substance to be immobilized is a ligand for affinity chromatography, the present invention can be used for purification of the substance, and when the substance to be immobilized is an enzyme having a catalytic action, bio- It is useful as a reactor, and in particular, the present invention has important significance in the technical field of biotechnology.

(Prior Art) There are many reports on a method of immobilizing a substance by a covalent bond method. Review articles include Toshiaki Osawa and Yoshio Terao, Affinity Chromatography and Affinity Labels, Protein Nucleic Acid and Enzymes, Supplement No. 22, 1980, Kyoritsu Shuppan.

The most frequently used method is to use a CNBr activated carrier to bind to the primary amino group of the substance to be immobilized (Axe
n, R., Porath, J., & Ernback, S. (1967): Nature 214 , 13
02.:Porath,J.,Axen,R., & Ernback, S. (1967): Nature
215 , 1941). This is a carrier with OH groups treated with CNBr,
This is a method of immobilizing by immobilizing an active imidocarbonate with a non-protonated primary amino group of the substance, but when the substance is a physiologically active substance such as an enzyme, many primary amino groups on the surface of the substance are used. Is bound to the carrier, the bond is strong, but on the other hand, it has a significant influence on the three-dimensional structure.
Physiological activity is greatly reduced. When the enzyme is immobilized by this method, the residual activity rate after immobilization is less than 10%.

Amino group of carrier and carboxyl group of substance to be immobilized,
Or vice versa, the carboxyl group of the carrier and the amino group of the substance to be immobilized are treated with a water-soluble carbodiimide (for example, N
-Ethyl-N '-(3-dimethyl aminopropyl) carbodi
imide hydrochloride, N-cyclohexyl-N'-2-
(4'-methyl-morpholinium) ethyl carbodiimide-
A method of immobilizing a substance on a carrier by binding it in the presence of p-toluene sulphonate) is also frequently used. However, this method also often greatly reduces the activity of the immobilized substance.

The present invention is a compound having two or more maleimide groups in one molecule, or a compound having both a maleido group and a succinimide ester group in one molecule, which is used to bind a substance to be immobilized with a carrier by a method. It is fixed. A compound having two or more maleimide groups in one molecule or a compound having both a maleimide group and a succinimide ester group in one molecule has been used for cross-linking formation of proteins.

Ishikawa et al. Have used it to form crosslinks between enzymes and antibodies, and many reports have been made.

As a review article (edited by Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, Enzyme-linked immunosorbent assay, 2nd edition, 1982, Ikusho Shoin), the method for binding an enzyme to an antibody is described in detail. However, all of these methods have been used for the cross-linking formation of soluble proteins, and the technique of firmly immobilizing the substance on the carrier without lowering the activity of the substance by such a method has never been known. Not new, new.

(Problems to be Solved by the Invention) In many of the known immobilization methods described above, the amino group of the substance to be immobilized is chemically bound to the carrier.
When the substance to be immobilized is a protein, the α-amino group at the amino terminus and the ε-amino group of lysine are often used for binding. These are highly reactive nucleophilic residues, but since they are present in large numbers in one molecule of protein, they are bound to the carrier at multiple points. Moreover, the positive charges of the α-amino group and ε-amino group play an important role in maintaining the three-dimensional structure of the protein, and the three-dimensional structure resulting from the loss of this positive charge due to immobilization The change in can't be ignored. Furthermore, when the substance is a physiologically active substance, it causes deactivation.

Also, when the carboxyl group of the substance to be immobilized and the carrier are chemically bound, almost the same problem occurs. When a protein is immobilized, there are β-carboxyl group of aspartic acid, γ-carboxyl group of glutamic acid, and C-terminal carboxyl group, which are present in large numbers in one molecule of protein. When this carboxyl group is used to bond to a carrier, many carboxyl groups are used for bonding to the carrier, so that the same problem as in the case of the amino group occurs.

In addition, in the other methods, when the carrier is epoxy-activated and the substance to be immobilized is made to act on it, the amino group, SH group, and OH group of the substance are bound, but this also has multiple points of substance and carrier. In many cases, the three-dimensional structure of the substance is affected by the binding, and the biological activity and the biological activity are greatly reduced.

In this way, the conventional method is to remove the substance to be immobilized and the carrier,
It can be said that the reaction group, which is present in large numbers in the substance to be immobilized, is bound to the carrier without considering which part of the substance to be immobilized is bound.

Originally, if the substance is firmly bound and fixed to the carrier, the activity is reduced accordingly. In other words, immobilization of binding and maintenance of activity are essentially incompatible. This point is not an exception even in the known methods described above, and in any method, if the substance is tightly bound, the decrease or disappearance of the activity is unavoidable, and in particular, the enzyme, the protein, various physiologically active substances, the immune-related substances, etc. It is no exaggeration to say that these methods cannot be used to fix substances.

(Means for Solving Problems) The present invention has been made to solve the above-mentioned drawbacks all at once, and includes a carrier binding method (covalent binding method, ionic binding method, physical adsorption method), and cross-linking. As a result of examining the immobilization method, the encapsulation method (lattice type, microcapsule type) and other known immobilization methods, it was concluded that the crosslinking method is the most suitable for achieving the object of the present invention. As a result of further deep and wide-ranging research from various fields, the present inventors found that 2
A compound having three or more maleimide groups or a compound having both a maleimide group and a succinimide ester group in one molecule is used. We found that the drop did not occur much, and completed the discovery.

The greatest feature of the present invention is that a compound having a maleimide group is used for immobilizing a substance. The present invention focuses on the point that the maleimide group specifically reacts with the SH group, and utilizes this. In the case of protein, SH only at cysteine residues
Although there are groups, many exist as cystines due to the cross-linking of peptide chains within proteins, and few free SH groups are present in the molecule. For example, in the case of bovine serum albumin,
There is only one molecule. Regarding other enzymes, etc., the amount of SH groups is smaller than that of amino groups and carboxyl groups. Therefore, it is considered that the substance immobilized on the carrier has less influence on the three-dimensional structure, and thus the activity of the substance is less likely to decrease.

As a matter of course, the method according to the present invention is not suitable for immobilizing a physiologically active substance having an SH group at the active center position (eg, SH protease).

However, many other physiologically active substances are useful immobilization methods for the above reasons.

Conventionally, as a method for immobilizing a substance having an SH group, a bond between an activated thiol carrier and an SH group-containing peptide is known as shown in the following reaction formula.

(Carlsson, J., Axen, R., and Unge, T. (1975) Eur.J.Bio
chemistry, 59 , 567) However, since the bond obtained by this method is a disulfide bond (-S-S-), it is unavoidable that the bond is easily dissociated by a reducing agent (such as glutathione) in the living body.

On the other hand, it is considered that the fixed substance bound by the thioether bond (-S-) is unlikely to dissociate with the reducing agent in the living body.

As described above, the immobilization method that does not adversely affect the physiological activity can be used as a high-performance bioreactor.

When the substance to be immobilized is a carrier of hapten which is an immunogen, particularly in the case of bovine serum albumin which is often used, albumin has 60 or more amino groups in one molecule and one molecule of It has an SH group. The hapten is often bound to the amino group or carboxyl group of albumin in order to bind to the carrier as much as possible.

In the thus-prepared hapten-carrier complex, when an amino group is used for binding, almost no free amino group is present, so the amino group cannot be used for binding to the carrier.

Therefore, it can be immobilized by binding to the carrier with an SH group that is not related to binding to the hapten. This is the reason why the method of the present invention is effective.

The hapten-carrier complex immobilized in this way can be analyzed by EIA (enzyme immunoassay) or RIA (radioimmunoassay).
Can be used as a solid-phased antigen. It can also be used as an affinity ligand for antibody purification.

Further, when immobilizing a ligand used for affinity chromatography, a large number of one-sided ligands are bound to a protein such as albumin, and this ligand-protein complex is bound to a carrier, whereby many ligands are bound to a fixed amount of the carrier. Can be introduced.

As shown in the formula, the higher the [L ₀ ] ligand concentration, the higher the K _D , and the carrier becomes suitable for affinity chromatography.

It is also considered that nonspecific adsorption is reduced by the effect of albumin and the like.

Further, as the compound which can be used in the method of the present invention and which has two or more maleimide groups in one molecule,
N, N '-(1,2-Phenylene) bismaleimide, N, N'-(1,3
−Phenylene) bismaleimide, N, N′− (1,4-Phenylen
e) bismaleimide, Azophenyldimaleimide, N, N′−Hexame
thylenebismaleimide, Bis (N-male-imidomethyl) et
Her is included.

Further, a compound having both a maleimide group and a succinylimide ester group in one molecule, which can also be used in the method of the present invention, is N-Succinimidyl.
-N-maleimidoacetate, N-Succinimidyl-4- (N-
maleimido) butyrate, N-Succimidyl-6- (N-malei
mido) hexanoate, N-Succinimidyl-4- (N-maleimi
domethyl) cyclo-hexane-1-carboxylate, N-Succin
imidyl-m- (N-maleimido) benzoate, N-Succinimi
dyl-p- (N-male-imidomethyl) -4-butyrate, N
-Sulfosuccinimidyl-4- (N-maleimidomethyl) cy
clohexane-1-carboxyl-ate, N-succinimidyl-m-
(N-maleimido) benzoate, N-sulfosuccinimidyl-p
-(N-maleimidomethyl) -4-butyrate and the like can be mentioned.

As the carrier, one having a polysaccharide skeleton (eg, agarose; Sepharose (manufactured by Pharmacia Fine Chemicals), dextran; Sephadex (manufactured by the same company), cellulose; (manufactured by Whatman)), one having a synthetic resin as a skeleton (eg , Polystyrene, polyacrylamide; Biogel P (manufactured by Bio-Rad), polyvinyl: Toyopearl (manufactured by Toyo Soda), glass or silica skeleton (eg, porous glass, silica gel, aminosilane derivative of porous glass) ), Natural products (eg Collodion,
Kaolin, alumina, carbon, bentonite, wool, wood). Furthermore, there are microbial cells, erythrocytes of various animals, and the like, and these carriers can introduce SH groups or amino groups as necessary.

For example, an SH group can be easily introduced into a carrier having an OH group as shown in the following reaction formula.

Further, when it is desired to introduce an amino group, ammonia may be allowed to act in the above step B as shown in the following reaction formula.

In addition to this, there are various methods for introducing an amino group or an SH group into a carrier, and an appropriate method can be selected as necessary.

The present invention is described below with reference to examples.

Example-1 Preparation of carrier a) Preparation of carrier having SH group To 90 g of wet weight Toyopearl HW-65, 90 ml of 1,4-butanediol glycidyl ether and 90 ml of 180 mg NaBH ₄ were prepared.
Add 0.6 M NaOH containing and shake for 8 hours in an incubator at 25 ° C. After the reaction, wash the carrier thoroughly with pure water, and
Add 0 ml of 1.3 M Na ₂ S ₂ O ₃ and shake at 25 ° C overnight. After shaking, adjust the pH to 7.0 with 0.1N hydrochloric acid. Wash the carrier again with pure water, add 270 ml of 25 mM dithiothreitol, and reduce at 37 ° C for 90 minutes. Then, it was washed thoroughly with 3M 0.1M sodium phosphate buffer containing 1 mM EDTA, pH 6.0, and S
Toyopearl HW-65 having H group was obtained. Product is 1g
Toyopearl has about 60 μmol SH group.

b) Preparation of carrier with amino group Toyopearl HW-65 of 90 g wet weight, 90 ml of 1,4-butanediol glycidyl ether and 90 ml of 180 mg NaBH ₄
Add 0.6 M NaOH containing and shake for 8 hours in an incubator at 25 ° C. After the reaction, wash the carrier thoroughly with pure water, and
Add 5 ml of concentrated ammonia water and shake at 40 ° C for 90 minutes. After shaking, the product was thoroughly washed with pure water to obtain Toyopearl HW-65 having an amino group.

Example 2 Immobilization of BSA (Bovine Serum Albumin) In 25 mg of BSA dissolved in 5 ml of 0.1 M phosphate buffer containing 1 mM EDTA and pH 6.0, N, N′-0-phenylenedi-maleimide2.
5 ml of the above-mentioned buffer solution containing 5 mg and 50 μl of dimethyl sulfoxide is added, and the mixture is reacted at 30 ° C. for 20 minutes. After the reaction, the excess N, N'-0 was applied to a 200 ml Sephadex G-25 column.
Excludes -phenylenedi-maleimide. 20 mg of the obtained maleimidated BSA and 2 g (wet weight) of the SH group-containing Toyopearl HW-65 obtained in Example 1 a) were mixed in the above buffer solution, and the mixture was mixed at 4 ° C. for 40 hours. Shake. The reaction solution was filtered through a Nutsche to obtain BSA immobilized on Toyopearl HW-65. Based on the difference between the amount of BSA that was not filtered and bound and the amount of BSA added, 12 mg of BSA could be immobilized per 2 g of the carrier.

Example 3 Immobilization of phosphorylcholine-BSA Phosphorylcholine is an affinity ligand specific to C-reactive protein, and phosphorylcholine is bound to BSA by reductive amination reaction.
Is. Phosphorylcholine-BSA was used instead of BSA, and the same as in Example 1 except for the above. 8 mg of phosphorylcholine-BSA could be immobilized per 2 g of carrier.

Example 4 Immobilization of β-galactosidase 5 mg of β-D-galactosidase (derived from Escherichia coli) and 1 mM EDT
0.5 g of N, N 'in 1 ml of 0.1 M phosphate buffer, pH 6.0 containing A
1 ml of the above buffer solution containing 0-phenylenedimaleimide and 10 μl of dimethylformamide was added and reacted at 30 ° C. for 20 minutes. After the reaction, the excess N, N'-0-phenylenedimale-imide is removed with a 40 ml Sephadex G-25 column. 4.2 mg of the obtained maleimidated β-D-galactosidase
And Toyopearl having the SH group obtained in Example 1 a)
Mix 2 g (wet weight) of HW-65 in the above buffer and mix at 4 ° C.
Shake for 18 hours. The reaction solution was filtered through a Nutsche to obtain β-galactosidase immobilized on Toyopearl HW-65. Due to the difference between the unbound β-galactosidase that was filtered and the added β-galactosidase, 3 g per 2 g of carrier.
8 mg could be immobilized. The activity was retained 82%.

Example-5 Immobilization of peroxidase 50 μM GMBS (N- (γ-maleimi was added to 1 ml of 0.1 M phosphate buffer pH 7.0 containing 6 mg of peroxidase (horseradish)).
100 μl of N, N-dimethylformamide containing dobutyloxy succinimide) is added and reacted at 30 ° C. for 30 minutes. After the reaction, add 40 ml of Sephadex G-25 column to the excess GMBS.
To remove. The maleimidated peroxidase obtained 5.
1 mg and 2 g (wet weight) of Toyopearl HW-65 having an SH group obtained in Example 1 a) were mixed in 5 ml of 0.1 M phosphate buffer pH 6.0 containing 1 mM EDTA, Shake at 4 ° C for 20 hours. The reaction mixture is filtered through a Nutsche and the Toyopearl HW-
A peroxidase immobilized on 65 was obtained. Filtered,
Due to the difference between unbound peroxidase and added peroxidase, 4.1 mg could be immobilized on 2 g of the carrier. The immobilized peroxidase retained 91% activity.

Example-6 Immobilization of phosphorylcholine-BSA Toyopearl having the amino group obtained in b) of Example-1
Equilibrate 2 g (wet weight) of HW-65 with 0.1 M phosphate buffer, pH 7.0. 50 μM EMCS (N- (ε-maleimidocap
2 ml of N, N'-dimethylformamide containing royloxy) succinimido) is added and reacted at 30 ° C for 30 minutes. The resulting maleimidated Toyopearl HW-65 contained 1 mM EDTA.
Equilibrate by washing with 0.1M phosphate buffer pH 6.0. 0.1M phosphate buffer pH 6.0 4 containing 10 mg of phosphorylcholine-BSA
Add ml and shake at 4 ° C for 48 hours. The reaction solution was filtered through a Nutsche to obtain phosphorylcholine-BSA immobilized on Toyopearl HW-65. The amount of unbound phosphorylcholine-BSA filtered and phosphorylcholine-B added
Based on the difference with SA, 3.1 mg of phosphorylcholine-BSA per 2 g of carrier
Was fixed.

Example 7 Immobilization of glucose oxidase In 0.1 ml of a 0.1 M phosphate buffer, pH 7.0 containing 10 mg of glucose oxidase (A. niger), 50 μM of GMBS (N- (γ-m
100 μl of N, N-dimethylformamide containing aleimidobutyloxy) succinimide) is added and reacted at 30 ° C. for 30 minutes. After the reaction, the excess GMBS is removed with a 40 ml Sephadex G-25 column. The obtained maleimidated glucose oxidase (8.2 mg) and 3 g (wet weight) of SH group-containing Toyopearl HW-65 obtained in Example 1 a) were mixed with 1 mM E
Mix in 0.1M phosphate buffer, pH 6.0, containing DTA at 4 ° C
Shake for 40 hours. The reaction solution was filtered through a Nutsche to obtain glucose oxidase immobilized on Toyopearl HW-65. 6.1 mg of glucose oxidase could be immobilized on 3 g of the carrier. The immobilized glucose oxidase is
It retained 73% activity.

(Effects of the invention) The present invention is a compound having two or more maleimide groups in one molecule or a compound having both maleimide groups and succinimide ester groups in one molecule, which binds a substance to be immobilized and a carrier. By adopting the completely new constitution of making it possible to firmly fix various substances to the carrier, the activity and other useful properties originally possessed by the substances are not damaged at all. Is played.

Therefore, the present invention, in particular, a substance whose activity is strongly reduced when firmly fixed, such as various proteins, enzymes,
It is particularly effective for immobilizing physiologically active substances, peptide drugs, antigens, antibodies, haptens, cells, cells and the like.

According to the present invention, since these substances are strongly bound to the carrier and do not easily come off, and further, there is no significant decrease in activity or destruction of the material, the following applications are possible. It can be widely used for: purification of substances, chemical production, biosynthesis, analysis, measurement, chromatography, dialysis, artificial organs, plasmapheresis, etc.

In particular, the present invention plays a very important role in each technical field of biotechnology, and the remarkable effects thereof are immeasurable.

Claims (10)

[Claims] 1. A method for immobilizing a substance, which comprises cross-linking a substance having an SH group and a carrier having an SH group with a compound having two or more maleimide groups in one molecule. 2. A substance having an SH group and / or an amino group,
A method for immobilizing a substance, which comprises cross-linking a carrier having an amino group and / or an SH group with a compound having both a maleimide group and a succinimide ester group in one molecule. 3. The method according to claim 1 or 2, wherein an insoluble carrier is used as the carrier. 4. The method according to claim 1 or 2, wherein the substance is a protein modified or not modified with a specific compound. 5. The method according to claim 4, wherein the protein is an enzyme. 6. The method according to claim 4, wherein the protein is albumin. 7. The method according to claim 4, wherein the specific compound is an affinity ligand or phosphorylcholine. 8. The method according to claim 5, wherein the enzyme is β-galactosidase. 9. The method according to claim 5, wherein the enzyme is glucose oxidase. 10. The method according to claim 5, wherein the enzyme is peroxidase.