JPH02111798A - Modified substance of protein - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula (Pro is protein or polypeptide; X is amino modifying group; L is carboxyl-modifying group, having >=3 amino groups and linked carboxyl group of Pro with one of the amino groups through amino bond: r is an integer having the number of amino groups of Pro in a nonmodified state as the upper limit; t is an integer between an integer of 1 to the number of the carboxyl groups of Pro in the nonmodified state as the upper limit). USE:A carrier for hapten used for hapten antibody action. PREPARATION:Amino groups of proteins are blocked under mild conditions and carboxyl groups are then amidated with a polyamine. A hapten is subsequently bonded through the carboxyl groups thereof to the introduced amino groups to afford the objective modified substance.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention can be used as a carrier for habten (low molecular weight antigen) used in producing habten antibodies, or as a carrier for producing multivalent hapten antigens used in various immunoassays. The present invention relates to modified protein or polypeptide modified products.

(Background of the invention) Antibodies (Ag) whose antigens (Ag) are low molecular weight compounds such as drugs
Ab) is produced by immunizing an immunized animal with a complex in which these low molecular weight antigens (habten) or their analogs are bound to a protein carrier such as BSA (bovine serum albumin). Among the functional groups present in the protein, an amino group or a carboxyl group can be considered as the habten binding site of the carrier protein, but conventionally habten has been mainly bonded to the amino group because the binding reaction is easy.

However, habten does not bind to the amino groups of protein carriers (the region where there are many unbound amino groups retains the protein's original three-dimensional structure, so it does not bind to the protein's original antigenic determinants). Therefore, antibodies (Ab") corresponding to the carrier protein itself are also produced in the serum obtained by immunization with the carrier protein.

For this reason, the obtained Habten antiserum (Ab) often needed to be previously absorbed by the carrier itself (Ag'').

Such disadvantages also occur when the same habten protein complex is used in so-called passive aggregation. Passive aggregation is a method in which a plurality of monoepitope habten (Ag) are supported on a carrier, and antigen/antibody binding occurs as an apparently multivalent antigen, thereby causing matrix aggregation. That is, a carrier carrying a plurality of Habten antigens (Ag) and an anti-Habten antibody (Ab) are brought into contact to form an antigen-antibody Madnox. The amount of formation is detected from changes in turbidity. At this time, if monovalent free Habten antigen coexists, matrix formation is inhibited and a decrease in turbidity is observed. From this decrease in turbidity, the amount of free hapten antigen, ie, the antigen in the measurement sample, can be quantified. In this case as well, if there is an antibody (Ab") for the carrier itself (Ag"), this Ag'-
Ab'' mediated matrix formation or aggregation occurs.

Therefore, the anti-hapten antibody (Ab) used here is either absorbed by the carrier itself (Ag") in advance, or passively absorbs a complex in which hapten (Ag) is bound to a carrier different from the carrier (Ag") used during immunization. It is necessary to subject it to an aggregation reaction system.

In order to eliminate this inconvenience, it is possible to block all the amino groups of the protein carrier with habten, thereby virtually completely denaturing the protein, but this would be extremely difficult to achieve, and even if it were possible, a large amount of habten would be removed. There was also the inconvenience that this method could not be used when there was only a trace amount of Habten.

On the other hand, if there is only a trace amount of habten, or if its solubility in a solvent is low (and only low-concentration habten solutions can be used), it may not be possible to incorporate enough habten into the carrier protein, and the resulting antibody titer will be insufficient. Therefore, it is desirable to increase the number of habuten introduction sites on the carrier as much as possible to provide a carrier with high habuten introduction efficiency.

(Objective of the Invention) The present invention was made in view of the above circumstances, and it is possible to almost completely modify and denature the amino groups of the carrier itself, so that antibodies of the carrier itself are not produced during immunization and passive aggregation is prevented. The first object of the present invention is to provide a protein modification/modification product that can carry habten on the same carrier without having to absorb anti-habten antibody (Ab) with the habten-carrying carrier itself (Ag'') in advance.

A second important objective is to provide a modified protein that not only does not substantially generate antibodies on the carrier itself, but also has a high efficiency of introducing Habten.

(Structure of the Invention) The first object of the present invention is achieved by a protein modification characterized by being represented by a structural formula.

Here, the protein in the protein modification product also includes a polypeptide, where Pro is a protein or a polypeptide; The amino group of one of the Pro
carboxyl modification group having an amide bond with the carboxyl group of; r is an integer with an upper limit of the number of amino groups possessed by unmodified Pro; t is an integer from 1 with an upper limit of the number of carboxyl groups possessed by unmodified Pro is an integer between the numbers.

Further, the second object of the present invention is achieved by a protein modification characterized by being represented by the structural formula (%).

In the formula, Pro is a protein or a polypeptide; Bonded carboxyl modification group; r is an integer whose upper limit is the number of amino groups possessed by unmodified Pro; t is an integer between the integer l and a number whose upper limit is the number of carboxyl groups possessed by unmodified Pro It is.

That is, the present invention blocks the amino groups of proteins (or polypeptides) with modifying groups, while blocking the carboxyl groups with 3
A new amino group is introduced by amidation with a polyamino or amino acid polymer having the above amino groups, and habten is bonded to the introduced amino group.

Furthermore, by selecting a reversibly releasable amino modifying group, this modifying group can be eliminated to regenerate the amino group of the protein, allowing habten to bind to both the regenerated amino group and the introduced amino group. This improves the introduction efficiency of hub ten.

Examples of blood proteins include BSA (bovine serum albumin),
KL)l (Keyhole Limped Hemocyanin)
, IgG, ferritin, thyroglobulin, etc. can be used, and natural or synthetic polypeptides can also be used as carriers.

Examples of the modifying group X that blocks the amino group of the carrier protein containing an ene and fi group include the following. First, the reversibly removable modifying groups are as follows.

-CO-0-R 5o2-R -CO-3-R 5-R -CO-Y-Z-R CHO -CO-CH2-COCH.

-CO-CF3 -CH, -C,H.

-CH-(C,H5)2C(C6Hs)- (wherein, R is a substituted or unsubstituted alkyl group, aryl group, or aralkyl group having 2 to 15 carbon atoms; Y is a substituted or unsubstituted methylene group; 2 is S or 0 atom; specific examples are given below.

-CO-0-R (urethane type amino protecting group); As the so-called urethane type amino protecting group such as alkyloxycarbonyl group (alkoxycarbonyl group), aryloxycarbonyl group and aralkyloxycarbonyl group, for example, t-butoxycarbonyl (Boc) group, inbutyloxycarbonyl group, benzyloxycarbonyl (Z) group, paramethoxybenzyloxycarbonyl group, t-amyloxycarbonyl group, d-inbornyloxycarbonyl group, adamantyloxycarbonyl group, and the like. Further, these may be partially substituted, such as a substituted benzyloxycarbonyl group having a halogen, nitro group, methoxy group, etc. introduced at the para position.

-SO□-R (sulfonyl type); 15Q2-Q-CH.

p-1-luenesulfonyl (Tosyl
) There is a group.

-CO-5-R: (phenylthio) carbonyl group (benzylthio) carbonyl group, (butylthio) carbonyl group, -3-R (thio type); 0-nitrophenylthio group o, p-dinitrophenylthio group -CO- Y-Z-R type; (Y is a substituted or unsubstituted methylene group: Z is S or 0
atoms) -CO-CH2-0-C,H5-No□0-nitrophenoxyacetyl group CH.

-CO-C-0-C6H, -NO2 CH.

0-Nitrophenoxyisobrobyl carbonyl group Others include CHO Formyl group -CO-CH2-COCH3 Acetoacetyl group -CO-CF3 Trifluoroacetyl group CH2 (
CM Ha ) benzyl group CH'-(C6H
5) 2 phenylmethyl group-”C(C8Hs)
3 triphenylmethyl (trityl) group -C=CH-R R2 (R, , R2 is an alkyl group), and the like.

All of the above amino protecting groups block the N atom of the amino group through a single bond, but the amino protecting group (-
NH2) may be blocked as in -N=CH-R to form a double bond with the N atom.

Introduction of these amino protecting groups and removal thereof can also be carried out according to known methods. For example, in the case of a urethane-type amino protecting group, the amino group can be blocked by an isocyanate method, an azidoformate method, a mixed carbonate method, a haloformate method, or the like. In addition, methods for removing the amino protecting group include catalytic reduction, Na-NHs treatment, acid treatment (
Examples include hydrogen bromide, trifluoroacetic acid, hydrogen fluoride, etc.), alkali treatment, electrolysis, and photolysis.

The above amino protecting groups, their introduction methods and removal methods are described in "Peptide Synthesis" (Izumiya et al., Maruzen, 1975).
"Basics and Experiments of Peptide Synthesis" (Izumiya et al., Maruzen, 1985) [Biochemistry Experiment Course 1. Chemistry of Proteins IVJ (edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1977)
try of Am1no Ac1ds”J, P, Gr
eenstein, M. Winitz, John
Wiley & 5ons, New York, Vo
l, II (19611"The Peptides E, 5chr6der, K. Li1bke, Ac
academic Press.

New York, vol. I, II (1
965) "Peptide 5 synthesis" M,
Bodanszky, M. A. Ondetti
Written by John Wiley & 5ons, New Yo
rk, (1966 & 1976)” Peptide
Chemistry 5eries.

Proceedings of the Japan
Symposium on Peptide C:he
mistry, 1976-1987Peptide
In5tituto, Protein Re5ea
rchFoundation, 0saka, Japan
Known techniques in peptide synthesis can be used, as described in detail in et al.

In addition, as a non-removable amino modifying group, C(CH2)m
There is an acyl group represented by CHa. However, m is an integer of O to 4, and by using an acyl group in this range, the water solubility of the protein modification product obtained can be ensured. Introduction of an acyl group into such an amino group can also be carried out according to the known techniques described in the above-mentioned known documents.

The number r of amino modification groups X introduced in this way is an integer that is the same as or smaller than the number p of amino groups possessed by the unmodified protein Pro.

The polyamino for amidating the carboxyl group of the carboxyl modifying group carrier protein is preferably a polyamino having 3 or more amino groups and 1 to 8 carbon atoms, and the water solubility of the protein modification obtained by using a polyamino in this range is Can be secured. In addition, the number of carbon atoms in the polyamino is selected, and the length between the carrier and the hubten (
spacer length) can be adjusted.

Here, the amino group is an amino group corresponding to primary amino (
-NH2) as well as an amino group corresponding to a secondary amino, a so-called imino group (>NH), and the polyamino described here is a concept that includes a polyimine having an imino group.

In other words, the amino group of the carboxyl-modifying group may be one that can bond to the carboxyl group of the carrier protein via an amide bond and also bond to the carboxyl group of habuten, and may be a primary amino group or a secondary amino group. It does not matter whether it is a class amino group or not.

Examples of such polyamino include the following.

diethylenetriaminoHzN-CH2CHz-NH-CH2CHz-NH2 triethylenetetramine H2N-(CH2CH2-NH) 2-CH2CH2-
NH2tetraethylenepentamineH2N-(CH2CH2-NHI 3-CH2CH2-
NH2pentaethylenehexamine H2N-fcH2cH2-NHI 4-Cl2CH2-
NH2 polyethylene polyamino H2N-(CH2CH2-NH) ll-CH2CH2
-NH2dipropylenetriaminoH2N-CH2CH2CH2-NH-(,H2(:H2
CH2-NH21,4-bis(aminopropyl)piperazinebis-hexamethylene-triaminoH2N-(CH2) 6-NH-(CH2) 6-NH
2 Furthermore, a polymer of amino acids (polyamino acids) having three or more amino groups may be used to amidate the carboxyl groups of the carrier protein. Examples of such amino acid polymers include polymers such as dimers and trimers of amino acids such as ornithine having α-amino and δ-amino, and lysine and oxylysine having a-amino and ε-amino. There is. Particularly preferred are lysylnosine and lysyllysyllysine, which are dimers and trimers of lysine, and ornithylornithine, which is a dimer of ornithine.

The number t of amino groups newly introduced into the carrier protein by the polyamino or amino acid polymer is at least 2, and the protein Pro in its natural state (that is, the state before amidation) is at least 2.
Maximize the integral multiple of the number of carboxyl groups S that has. Taking into consideration that the amount of a normal habten protein complex introduced is usually 10 molecules/carrier or more, preferably 20 molecules/carrier or more, the number of amino groups introduced is usually 10 or more, preferably 20 or more.

Therefore, if the aforementioned amino modifying group X is almost completely eliminated,
The number of amino groups on the modified protein is usually 10 or more, preferably 20 or more, more than that of the unmodified protein, resulting in a higher efficiency of introduction of habuten.

In addition, since there are two or more amino groups in the modification group introduced into the carboxyl group of l, the number of points for introducing habten is further increased, and further improvement of habten introduction efficiency can be expected. When the tertiary structure of a high molecular weight molecule changes due to blocking by an amino modifying group, even if the modifying group is removed, the high molecular weight molecule does not easily return to its original higher order structure and remains denatured. Therefore, even if the modifying group is removed from a protein with more blocked amino groups, it will substantially no longer produce antibodies against the carrier itself, which would occur in an unmodified state.

Next, the method for producing the modified protein of the present invention will be explained.

As shown in the flowchart of FIG. 1, the amino groups of the protein are first blocked with a modifying group that can be removed under mild conditions (step 1). In the case of a urethane-type amino modification group, the amino group can be almost completely blocked by using a modifier in an amount several times the molar amount of the amino group, usually under weak alkalinity.

Next, the carboxyl group is amidated with polyamino (step 2). As the polyamino, diethylenetriamino, triethylenetetramine, etc. are used. The reaction is carried out by activating the carboxyl group of the protein with carbodiimide and adding it to a large excess amount of polyamino (an excess amount such that intramolecular crosslinking and intermolecular crosslinking will not substantially occur).

Note that since most of the protein-derived amino groups are blocked in Step 1, intra- and intermolecular cross-linking of the carrier does not occur during the activation of carboxyl groups in Step 2. If step 2 is performed without performing step 1, the carrier will polymerize due to intermolecular crosslinking, resulting in a non-uniform modified product. In addition, the solubility in water generally decreases greatly, making it difficult to carry out the reaction for introducing habuten. In this regard, according to the present invention, since there is no intramolecular or intermolecular crosslinking of the carrier, it is possible to obtain a modified protein for a carrier that is uniform and has good water solubility.

In the protein modification product thus obtained, most of the protein-derived amino groups are blocked, and only the newly introduced polyamino-derived amino groups are present in the protein.

By bonding habten to this introduced amino group via its carboxyl group, a habten-carrying carrier is obtained, which can be used to immunize an immunized animal (step 4).

At this time, since habten is not necessarily bonded to all of the introduced amino groups, the number i of habten introduced is smaller than the number t of introduced amino groups. However, it is usually about 10 or more.

On the other hand, when the above-mentioned amino modifying group Both of these will exist. Step 1, 2
If the protection and removal of amino groups is ideally performed, the number of regenerated amino groups (11) will be restored to approximately the same number as the original amino groups p of the unmodified protein. In comparison, there are approximately as many amino groups as an integral multiple of the number t of amino groups introduced into carboxyl groups (several times the number of amino groups in the polyamino used minus 1). Therefore, the number of reaction points for introduction of habten can be increased to the maximum, allowing efficient introduction of habten, and a habten carrier conjugate suitable for producing high-titer antibodies can be obtained (step 5).

Even in this case, habuten does not bind to all amino groups, and the number of habuten that binds to the regenerated/introduced amino groups j
and k are smaller than the number of regenerated amino groups and the number of introduced amino groups t, respectively, but the total number of amino groups (h + t) is larger than when using an unmodified protein or a carrier obtained in step 2 that blocks the original amino groups of the protein. Therefore, the efficiency of introducing hub ten is high. It is said that the number of habten introduced for immunity is usually 10 or more per carrier molecule, but with protein modifications that maximize the habten introduction points as in the present invention, this number can be easily exceeded. .

Furthermore, if the modified protein according to the present invention is used as a carrier to support multiple Habten antigens (Ag) by passive aggregation,
Since the protein has been sufficiently denatured by the modification reaction in step 1 and has lost its antigenicity to the original protein, even if it is brought into contact with a carrier carrying multiple anti-Habten antibodies (Ab), the antibodies (Ab) will not react with the carrier itself ( Therefore, there is no need to absorb the antiserum (Ab) used in advance with the carrier itself (Ag''), and there is no need to use a carrier other than the carrier used here.

This also applies when the amino group is regenerated in step 3. In other words, low-molecular-weight peptides can completely return to their original structure by removing the amino-modifying group, but in high-molecular-weight molecules such as proteins and polypeptides, once the tertiary structure changes due to blocking by the amino-modifying group, even if Even if the protecting group is removed, the structure remains denatured without returning to its original higher order structure. Therefore, even if the regenerated amino group is used as a point for introducing habten, antibodies of the carrier itself, which would occur in an unmodified state, are not substantially generated.

(Effects of the Invention) As described above, the first invention blocks the amino groups of proteins with a removable or non-removable modifying group, while amidating carboxyl groups with polyamino or polyamino acids to create new amino groups. introduced.

Therefore, the degree of denaturation of the protein can be increased by eliminating or reducing the amino groups derived from the protein, and there is substantially no generation of antibodies (Ab'') against the carrier protein itself.

In addition, since two or more amino groups are introduced into the carboxyl group of l, there are many points for introducing habten, and further improvement in habten introduction efficiency can be expected. Therefore, antibodies (Ab) can be produced efficiently even when habten is in a small amount. can do.

Also, when used for passive agglutination, anti-Habten antibody (Ab)
It is not necessary to absorb the antigen in advance with the carrier itself (Ag''), and a multivalent hubten antigen complex can be created using the same carrier used during immunization.

In addition, in the second invention, the blocked amino group is regenerated so that both the regenerated amino group and the introduced amino group can bind to the habuten, so there are more habuten binding sites than the unmodified protein, and the habuten is improved. High implementation efficiency. Therefore, antibodies (Ab) can be efficiently produced even with a trace amount of Habten.

Furthermore, since it has been modified once to protect amino groups, the degree of denaturation is high (and the carrier itself does not substantially generate antibodies (Ab'')).

(Example) This will be explained below using examples.

Example 1-1; Preparation of acetylated BSA BSA (manufactured by Sigma) Ig was added to 100 ml of 50 mM phosphate buffer (p
H9), and 0.5 ml of acetic anhydride was added dropwise thereto under water cooling. During the dropping, monitor the pH and add 1N NaO.
The pH was maintained at 9 with H solution. After completion of the dropwise addition, the mixture was reacted for 30 minutes at room temperature with stirring. The reaction solution was gel-filtered and desalted through a Sephadex G-10 (manufactured by Pharmacia) column equilibrated with 1% acetic acid to separate acetylated BSA. The yield after freeze-drying was 1.05 g.The number of remaining amino groups after the reaction was determined by fluorescamine
Mine; manufactured by F-Hoffmann U. Rocher), the amount was about 0.1% of the BSA before the reaction.

Example 1-2 Amino Group in Niacetylated BSA A new amino group was introduced by condensing the carboxyl group of acetylated BSA with diethylenetriamino.

1 g of acetylated BSA prepared in Example 1-1 was added to 50 m
1 of 50mM phosphate buffer (pH 8), and to this was added 5ml of a 5% aqueous solution of water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide).
1 was added dropwise to the mixture under water cooling to activate the carboxyl group of the Boc-conjugated BSA. The aqueous solution was added dropwise to 50 ml of IM diethylenetriamino aqueous solution (pH 8) with stirring under water cooling and mixed to react. After returning to room temperature and reacting for 1 hour, the reaction solution was dialyzed and desalted against running water, and then freeze-dried. When the amount of introduced amino groups in the obtained sample was quantified using fluorescamine, it was found that about 50 molecules of new diethylenetriamino were introduced per molecule of B5Al.

1-Carpoxybutyrulfenobarbicur was conjugated with BSA modification as habuten.

100 mg of BSA modified product obtained in Example 2-2 was added to 10 ml
was dissolved in 50mM Tris-HCl buffer (pH 9). On the other hand, ■-Carboxybutyrulfenobarbicur 100
Dissolve mg in 5 ml of dimethyl sulfoxide and add 1
1 equivalent of triethylamino and 1.1 equivalent of isobutylchloroformic acid were added to form a mixed acid anhydride and activated.

This mixed acid anhydride solution was added to the above BSA modification solution and reacted. After reacting at 4° C. for 30 minutes and further at room temperature for 1 hour, the reaction solution was dialyzed against running water to desalt. For the sample after freeze-drying, the amount of phenobarbicool introduced was determined by ultraviolet absorption spectrum (280 nm and 300 nm).
(measured by wavelength). The amount introduced was as high as 67 mol per B5Al molecule.

When rabbits were immunized with the obtained habten-carrying BSA according to a conventional method, a high titer anti-phenobarbital serum was obtained.

Procedure 2-1: Preparation of Boc-modified BSA The amino groups of BSA were blocked by converting them into t-butoxycarbonyl (Boc) by a mixed carbonate method.

BSA (manufactured by Sigma) Ig, 100ml of 50mM
Dissolve in phosphate buffer (pH 9), add 0.6 ml of triethylamino, and add 50 ml of a 2% dioxane solution of t-butyl (4,6-dimethylpyrimidyl-2-thiol) carbonate (manufactured by Aldrich). Mixed dropwise. After stirring the reaction overnight at room temperature, add 100 ml of water.
was added to stop the reaction. After extracting and removing unreacted carbonate with ethyl acetate, the aqueous layer was dialyzed against running water to desalt. The yield of Boc-conjugated BSA after freeze-drying is 1.0
It was 8g. When the number of amino groups remaining after the reaction was determined using fluorescamine, it was found to be 0.1% or less of the BSA before the reaction.

Example 2-2: Induction of amino group into Boc-modified BSA A new amino group was introduced by condensing the carboxyl group of Boc-modified BSA with diethylenetriamino.

50ml of 1g of Boc-based BSA prepared in Example 2-1
of 50 mM phosphate buffer (pH 8), and add 5 ml of a 5% aqueous solution of water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) to this solution.
were mixed dropwise under water cooling to activate the carboxyl group of Boc-conjugated BSA. The aqueous solution was added dropwise to 50 ml of a 1M diethylenetriamino aqueous solution (pH 8) with stirring under water cooling and mixed to react. After returning to room temperature and reacting for 1 hour, the reaction solution was dialyzed and desalted against running water, and then freeze-dried. When the amount of introduced amino groups in the obtained sample was quantified using fluorescamine, it was found that about 55 molecules of new diethylenetriamino were introduced per molecule of B5A.

(2)-Carpoxybrovirulfenobarbicur was combined with BSA modification as habuten.

100 mg of BSA modified product obtained in Example 2-2 was added to 10 ml
of 50 mM Tris-HCl buffer (p) (9).

On the other hand, 1-carpoxybrobyrufuenobarbital 1
00 mg was dissolved in 5 ml of dimethyl sulfoxide, and 1.1 equivalents of triethylamino and 1.1 equivalents of isobutylchloroformic acid were added to form a mixed acid anhydride and activated. This mixed acid anhydride solution was added to the above BSA modification solution and reacted. After reacting at 4°C for 30 minutes and further at room temperature for 1 hour, the reaction solution was dialyzed against running water to desalt. Regarding specimens after freeze-drying.

The amount of phenobarbicur introduced was determined by the ultraviolet absorption spectrum (28
'On m and 300 nm). The amount introduced was as high as 65 mol per B5Al molecule.

When rabbits were immunized with the obtained habten-carrying BSA according to a conventional method, a high titer anti-phenobarbital serum was obtained.

Example 3-1: Example 2 of amino protecting group of BSA modified product
50' Om g of the modified BSA obtained in step 2 was dissolved in 50 ml of hydrogen bromide/glacial acetic acid, and reacted at room temperature for 20 minutes.

After the reaction, the solvent was distilled off under reduced pressure, 100 ml of water was added, and this was desalted by dialysis with running water, and then freeze-dried. The obtained sample weighed approximately 440 mg. When the amino group was quantified using fluorescamine, it was about 1 per B5Al molecule.
15 amino groups were detected, and the difference from the value of about 55 in Example 2 showed that l l 5-55m60 amino groups were regenerated.

The files were combined. The amount of phenobarbicool introduced per B5Al molecule was 83 mol, which was higher than the amount introduced into the carrier before amino group regeneration (Example 2-3: 65 mol).

When rabbits were immunized with the obtained hapten-carrying BSA,
High titer anti-phenobarbital serum was obtained.

[Brief explanation of drawings]

FIG. 1 is a flowchart illustrating a method for producing and using a modified protein according to the present invention. Patent applicant: Fuji Photo Film Co., Ltd. Agent: Patent attorney: Yama 1) Text: Yama 1) Hiroshi
capital

Claims (11)

[Claims] (1) Structural formula; ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, in the formula, Pro is a protein or polypeptide; a carboxyl modification group which has an amide bond with the carboxyl group of Pro through one of the amino groups; r is an integer whose upper limit is the number of amino groups that Pro has in an unmodified state; t is an integer from 1 to a non-carboxyl modification group; 1. A modified protein, which is an integer between the upper limit of the number of carboxyl groups possessed by Pro in a modified state. (2) The amino modification group X is ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (However, m is an integer from 0 to 4) The protein modification product according to claim 1, characterized in that it is. (3) The protein modification product according to claim 1, wherein the amino modification group X is selected from the following structural formula. -CO-O-R -SO_2-R -CO-S-R -S-R ▲There are mathematical formulas, chemical formulas, tables, etc.▼ ▲There are mathematical formulas, chemical formulas, tables, etc.▼ -CHO -CO-CH_2-COCH_3 -CO -CF_3 -CH_2-C_8H_5 -CH-(C_6H_5)_2 -C-(C_6H_5)_3 (However, R is a substituted or unsubstituted alkyl group, aryl group, or aralkyl group having 2 to 15 carbon atoms) (4) The amino modifying group basis,
8. The protein modification product according to claim 7, wherein the modified protein is an aryl group or an aralkyl group. (5) Protein modification according to any one of claims 1 to 4, wherein the carboxyl modification group L is a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms and having 3 or more amino groups. thing. (6) The protein modification product according to any one of claims 1 to 4, wherein the carboxyl modification group L is a polymer of amino acids having two or more amino groups. (7) Structural formula; ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, in the formula, Pro is a protein or polypeptide; a carboxyl modification group having an amide bond with the carboxyl group of Pro through one of the amino groups; r is an integer whose upper limit is the number of amino groups possessed by unmodified Pro; h is an integer from 1 to p; t is an integer between the integer 1 and the upper limit of the number of carboxyl groups possessed by unmodified Pro. (8) The protein modification product according to claim 7, wherein the amino modification group X is selected from the following structural formula. -CO-O-R -SO_2-R -CO-S-R -S-R ▲There are mathematical formulas, chemical formulas, tables, etc.▼ ▲There are mathematical formulas, chemical formulas, tables, etc.▼ -CHO -CO-CH_2-COCH_3 -CO -CF_3 -CH_2-C_6H_5 -CH-(C_6H_5)_2 -C-(C_6H_5)_3 (However, R is a substituted or unsubstituted alkyl group, aryl group, or aralkyl group having 2 to 15 carbon atoms) (9) The amino modifying group basis,
8. The protein modification product according to claim 7, wherein the modified protein is an aryl group or an aralkyl group. (10) Protein modification according to any one of claims 7 to 9, wherein the carboxyl modification group L is a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms and having 3 or more amino groups. thing. (11) The protein modification product according to any one of claims 7 to 9, wherein the carboxyl modification group L is a polymer of amino acids having two or more amino groups.