JPH07121222B2 - Chemically modified serine protease-containing composition, its production method and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a chemically modified serine-protease-containing composition having substantially no catalytic activity, but having a novel ligand activity, a production method, and the composition. It has various uses for medicine and industry, and is particularly useful for separation, affinity purification, diagnostic agents and the like.

[Conventional technology]

1960 BS Hartley (Animal
Review of Biochemistry (Annu.Rev.Bioch
em.) Vol. 29, p. 45 (1960), various proteases belonging to serine proteases have been discovered since the proteases were classified according to the nature of the active group. Among them, 1969 DM Blow et al. (Nature, Vol. 221, p. 337 (1969)) showed that the active centers were Ser-195 and His-5 based on the results of X-ray analysis of chymotrypsin.
7.Clarifying that Asp-102 has a hydrogen-bonded structure, advocating the importance of these three amino acid residues in the catalytic activity, many similar reports have been made for other serine proteases. .

On the other hand, although there are many reports on the scientific modification of serine proteases, there are few reports that chemically modified serine proteases exhibit activity as novel affinity ligands. S. Ishii et al. [Biological and Biochemical Research Communities <Biological and Biochemical Research Communicat
ions> Vol. 72, p. 1443 (1976) and Journal of Biochemistry (J. Biochem.) Vol. 81, p. 647 (1977)], but there are reports on other serine proteases. Absent. That is,
Anhydrotrypsin obtained by PMSF (phenylmethanesulfonyl fluoride) and alkaline treatment is the only report that it has a high affinity for peptides whose C-terminal amino acid is Arg or Lys.

[Problems to be solved by the invention]

In the method of Ishii et al., That is, a method in which salmin sulfate is digested with trypsin, the digest is immobilized on a carrier, and anhydrotrypsin produced by PMSF and alkali treatment is purified by the immobilized column, the operation is It ’s complicated,
In addition, the yield is poor and it is not suitable for industrial production.

In addition, since the chemically modified trypsin produced by the above method contains unmodified trypsin, the trypsin activity remains 0.1% before the chemical modification, and the chemically modified trypsin composition is immobilized to separate and purify the peptide. In that case, the peptide was fragmented due to the remaining trypsin activity, which was a problem in terms of application.

An object of the present invention is to provide a chemically modified serine protease-containing composition having affinity ligand activity but substantially no catalytic activity, and a method for producing the same and use thereof.

[Means for solving problems]

Briefly describing the present invention, the first invention of the present invention provides a composition containing a chemically modified serine protease, wherein a serine protease containing chemically modified serine at the active center is treated with a non-chemically modified serine protease. It has an affinity ligand activity characterized by being modified with a compound that acts only on active center histidine, and then fractionated by using affinity ligands having different affinities depending on the presence or absence of chemical modification of active center histidine. It relates to a chemically modified serine protease-containing composition having substantially no activity.

The second invention of the present invention also relates to a method for producing the composition of the first invention, which comprises a serine protease chemically modified at the active center serine, which has an affinity ligand activity but substantially no catalytic activity. In a method for producing a composition, a serine protease-containing crude product obtained by chemically modifying active center serine is modified with a compound that acts only on active center histidine of unmodified serine protease, and then chemical modification of active center histidine. It is characterized by performing separation using affinity ligands having different affinities depending on the presence or absence of.

The third invention of the present invention relates to the use of the composition of the first invention, which comprises one or two chemically modified serine proteases.
In the method for fractionating peptides using more than one species, the chemically modified serine protease-containing composition of the first invention of the present invention is used as the serine protease.

The present invention relates to chemically modifying a serine protease, and the resulting chemically modified serine protease has substantially no catalytic activity, but expresses a novel affinity ligand activity. For example, such a chemical modification method according to the present invention includes a chemical modification to an amino acid residue in the active center of serine protease. Such chemical modification methods include specific chemical modifications to the serine residue and histidine residue in the active center. Further, the method for purifying the chemically modified serine protease relates to a method using an affinity ligand such as an immobilized protease inhibitor.

It has been found that the chemically modified serine protease containing compositions of the present invention do not substantially retain protease activity.

On the other hand, the chemically modified trypsin prepared by the conventional method retains a large amount of trypsin activity due to unmodified trypsin, resulting in functional heterogeneity.

In a preferred embodiment of the present invention, it was shown that the chemically modified serine protease shows high specificity for the reaction product cleaved by the serine protease, and becomes a novel affinity ligand.

The present invention further includes in vivo or in vitro.
In vitro), the use of the chemically modified serine protease-containing composition as an affinity carrier for isolating, purifying, and measuring a peptide fragment generated by cleavage with a serine protease is also included.

When it is desirable to convert the amino acid residue at the active center into another amino acid in chemically modifying serine protease in the present invention, an amino acid substitution method by a genetic engineering method is used instead of a chemical or enzymatic modification method. The modification method used may be used.

Hereinafter, the present invention will be specifically described.

Selection of Serine Proteases According to the present invention, serine proteases with any substrate specificity may be used. However, it is desirable to use a serine protease having a clear substrate specificity and a narrow substrate specificity. This is because it facilitates the analysis of the results in the separation, purification, and measurement of peptide fragments generated by cleavage with serine proteases, and by combining several serine proteases, very useful information can be obtained. Is. Examples of serine proteases used in the present invention are listed below.

Trypsin, pronase, thrombin, urokinase,
Enterokinase, acrosin, plasmin, kallikrein, collagenase, chymotrypsin, cathepsin G,
Lysyl endopeptidase, carboxypeptidase Y, subtilisin, cucumicin, elastase, proline specific protease, blood coagulation protease.

Production of Chemically Modified Serine Perotease-Containing Composition The production of the chemically modified serine protease-containing composition of the present invention can be carried out, for example, by first modifying the serine residue of the serine protease and then modifying the histidine residue. . As the compound that modifies the histidine residue, a compound that does not act on the active center serine is used. By this method, a chemically modified serine protease-containing composition having no catalytic activity, but having novel affinity ligand activity can be prepared, and its purification can be facilitated. Separation and purification of the crude product containing the chemically modified serine protease is performed using affinity ligands having different affinities depending on whether or not the active center histidine is chemically modified. For example, a carrier on which an inhibitor of serine protease is immobilized can be used.

Table 1 shows examples of the serine residue-modifying reagent and histidine residue-modifying reagent used in the present invention, and further examples of immobilized inhibitors.

Immobilization of Chemically Modified Serine Protease-Containing Composition on Carrier A chemically modified serine protease-containing composition is formed by using a conventionally known method for immobilizing a protein on an insolubilized carrier. Examples of the insolubilized carrier include BrCN activated sepharose 4B (manufactured by Pharmacia).

Uses of Chemically Modified Serine Protease-Containing Composition The method of using the chemically modified serine protease-containing composition of the present invention includes specific separation / separation of protease digests in the fields of genetic engineering, protein engineering, biochemistry, clinical diagnosis and the like.
Purification can be mentioned.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Composition containing immobilized chemically modified trypsin 1) Chemical modification of trypsin 200 mg of bovine-trypsin (manufactured by Sigma) was added to 0.05 M Tris-HCl buffer pH 7.0 (containing 0.02 M CaCl ₂ ) 20
35 mg of PMSF (manufactured by Sigma) dissolved in 0.2 ml of acetone is added, and the mixture is stirred at 25 ° C for 30 minutes. This time,
Keep the pH at 7.0 with 0.02M NaOH. P with 1M HCl
The reaction is stopped by setting H to 3 or less. This reaction solution was added to 1 mM HC
Dialyze overnight at 4 ° C. To this is added 1 / 19th volume of 1M KOH at 0-2 ° C., left for 10 minutes, then adjusted to pH 7 with acetic acid. Finally pH 7.0, acetic acid concentration 0.0
Adjust the 5M Ca ²⁺ concentration to 0.02M. TLCK this solution at room temperature
(Manufactured by Sigma) was added to a final concentration of 200 μM, and 1
After reacting for 50 minutes, 50 mM Tris-HCl buffer pH 8.0 (0.5 M
It was dialyzed against NaCl (containing NaCl) overnight at 4 ° C. and then subjected to p-aminobenzamidine agarose (manufactured by Pierce). 45.4 mg of a chemically modified tribucin-containing composition was obtained which was eluted with 50 mM Tris-HCl buffer pH 8.0 (containing 0.5 M NaCl) and then eluted with 10 mM HCl (containing 0.5 M NaCl).

In addition, the trypsin activity of this fraction was adjusted to 1 mM Bz-Arg-pNA.
Using (Peptide Laboratory Ltd.) to measure the increase in the 410nm absorbance in 50mM Tris -HCl Batsufa -pH8.2 (20mM CaCl ₂ containing). As a result, the residual trypsin activity was 0.01% or less of the trypsin activity before chemical modification.

2) Immobilization of chemically modified trypsin-containing composition on sepharose 4B Chemically modified trypsin-containing composition 10 obtained by the above method
mg was immobilized on 2 ml of BrCN-activated Sepharose 4B (manufactured by Pharmacia) according to the user guide of Pharmacia. The residual trypsin activity of the obtained composition containing immobilized chemically modified trypsin (2 ml) was 50 mM Tris-HCl pH8.2 (containing 20 mM CaCl ₂ ) using 1 mM Bz-Arg-pNA as a substrate.
The increase in absorbance at 410 nm was measured in a buffer. As a result, it was less than 0.001ΔA410 / min / mol gel. Also, B
The adsorbed amount of z-Gly-Arg (manufactured by Peptide Institute) was measured as follows. Start buffer (50 mM acetate buffer
250n on gel (bed volume 1ml) equilibrated with pH 5.0)
After adding the mol of Bz-Gly-Arg and removing the unadsorbed fraction with a starting buffer, the adsorbed Bz-Gly-Arg was removed with 0.1 M HCOOH.
It was eluted at (pH 2.5). Bz-Gly-Arg was quantified by the Sakaguchi reaction.

As a result, the adsorption capacity of the immobilized chemically modified trypsin-containing composition gel was 60 nmol Bz-Gly-Arg / ml gel.

The column operation was performed at 4 ° C.

The composition containing immobilized chemically modified trypsin prepared as described above was stably stored in 50 mM acetate buffer (pH 5.0) containing 20 mM CaCl ₂ and 0.05% NaN ₃ .

3) Novel Affinity Ligand Activity of Immobilized Chemically Modified Trypsin-Containing Composition The novel affinity ligand activity of the immobilized chemically modified trypsin-containing composition prepared by the above method was examined by the following method.

3-1) Fractionation of Nieurotensin and Tryptic Digestion Product of Nieurotensin by Chemically Modified Trypsin-Containing Composition Column Immobilization Gel was filled in the column to make a column of 6.3 ml,
Double column volume starting buffer (50m containing 20mM CaCl ₂
Equilibrate with M acetate buffer pH 5.0). Niurotensin (88n mol / 0.8ml: Peptide Research Laboratories) and 310uromol of niurotensin with 1nmol trypsin in 1ml of 20mM borate buffer pH 8.0 (containing 20mM CaCl ₂ ) at 25 ° C 5.
The solution digested for 5 hours was diluted with a starting buffer so that the final concentration was 100 nmol / ml, and applied to each column.

Wash the column with about 20 column volumes of starting buffer. The gel bound fraction is then eluted with 5 mM HCl (pH 2.5). Fractions were collected in 3 ml aliquots and all operations were carried out at 4 ° C. The peptide was detected by measuring the guanidino group by the ninhydrin / fluorescence method. The results are shown in Fig. 1.
That is, FIG. 1 is a graph showing the novel affinity ligand activity of the immobilized chemically modified trypsin-containing composition of the present invention, and FIG. 1-1 is a graph showing the elution pattern when Nieurotensin is added. 1 and 2 are graphs showing the elution pattern when a trypsin digestion product of niurotensin was added. The arrow indicates the elution buffer is 5 m.
It shows the time when it was changed to M HCl (pH 2.5), the horizontal axis is the number of fractions,
The vertical axis represents fluorescence intensity. As a result of analyzing each peptide fraction with a peptide sequencer, the pass-through peptide fraction in Fig. 1-1 was Pyr-Leu-Tyr-Glu-ASn-Lys-Pro-Arg-Ar.
It was g-Pro-Tyr-Ile-Leu. In addition, the flow-through and adsorbed peptide fractions in Fig. 1-2 are respectively Arg-Pro-Tyr-I.
le-Leu and Pyr-Leu-Tyr-Glu-ASn-Lys-Pro-Arg
And corresponded to the C-terminal peptide fragment and the N-terminal peptide fragment.

From the above results, it was shown that the chemically modified trypsin-containing composition of the present invention specifically adsorbs a peptide having less than C amino acid Arg, and it was confirmed that the composition exhibits novel affinity ligand activity.

Example 2 Fixed morphology-modified lysyl endopeptidase-containing composition 1) Chemical modification of lysyl endopeptidase Lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) 200 ml
Was used in the same manner as in Example 1 to prepare a chemically modified lysyl endopeptidase-containing composition. As a result, 51.3 mg of a chemically modified lysyl endopeptidase-containing composition was obtained. Also,
The chemically modified lysyl endopeptidase activity was determined to be 2.5 mM Bz
-Lys-pNA (manufactured by Peptide Institute) and 0.2 M Tris-
The increase in absorbance at 410 nm was measured in HCl buffer (pH 9.0). As a result, the residual lysyl endopeptidase activity was 0.001 of the lysyl endopeptidase activity before chemical modification.
It was less than 5%.

2) Immobilization of chemically modified lysyl endopeptidase-containing composition on cepharose 4B 10 mg of the chemically modified lysyl endopeptidase-containing composition obtained by the above method was added to 2 ml BrCN-activated cepharose 4B.
(Farmassia). Bz-Gly of the obtained composition containing immobilized chemically modified lysyl endopeptidase
The adsorption capacity of -Lys (manufactured by Peptide Institute) was measured as follows. Start buffer (50 mM acetate buffer pH 5.
250 nmol on a gel (bed volume 1 ml) equilibrated with (0)
Bz-Gly-Lys was added, and the unadsorbed fraction was eluted with a starting buffer, and the adsorbed Bz-Gly-Lys was washed with 0.1 M HCOOH (p
H2.5). Bz-Gly-Lys was quantified by measuring the fluorescence intensity with fluoresamine (manufactured by Sigma). As a result, the adsorption capacity of the composition gel containing the immobilized chemically modified lysyl endopeptidase was 70 nmol Bz-Gly-Lys / ml gel.

The composition containing immobilized chemically modified lysyl endopeptidase prepared as described above was stably stored in 50 mM acetate buffer (pH 5.0) containing 0.05% NaN ₃ .

3) Novel Affinity Ligand Activity of Immobilized Chemically Modified Lysyl Endopeptidase-Containing Composition 3-1) Immobilized Chemically Modified Lysyl End of Lysyl Endopeptidase Digestion Product of β-Neo Endorphin and β-Neo Endorphin Fractionation by peptidase-containing composition column Fill the column with a gel to make a 6.3 ml column, and
Double column volume starting buffer (50 mM acetate buffer pH
Equilibrate with 5.0). β-Neo Endorphin (90n mo
l / 0.9 ml: manufactured by Peptide Institute) and 400 nmol of β-neoendorphin at 10 nm with 1 nmol of lysyl endopeptidase.
In 1 ml of mM Tris-HCl buffer (pH 9.0) at 37 ° C, 6
The solution digested for a time was diluted with a starting buffer so that the final concentration was 100 nmol / ml, and each was applied to a column. 20 columns
Wash with double column volume starting buffer. The gel bound fraction is then eluted with 5 mM HCl (pH 2.5). Fraction 3
ml was collected, and all operations were performed at 4 ° C. The peptide was detected by measuring the fluorescence from fluoresamine. The results are shown in Fig. 2. That is, FIG. 2 is a graph showing the novel affinity ligand activity of the composition containing the immobilized chemically modified lysyl endopeptidase of the present invention, and FIG. 2-1 shows that when β-neoendorphin was added. 2-2 is a graph showing the elution pattern when β-neoendorphin was digested with a lysyl endopeptidase. The arrow, the horizontal axis, and the vertical axis have the same meanings as in FIG.

As a result of analyzing each peptide fraction with a peptide sequencer, the flow-through peptide fraction of Fig. 2-1 was Tyr-Gly-Gly.
-Phe-Leu-Arg-Lys-Tyr-Pro. Also, the second
-The flow-through and adsorbed peptide fractions in Figure 2 are Try-Pro and
And Tyr-Gly-Gly-Phe-Leu-Arg-Lys, which corresponded to the C-terminal peptide and the N-terminal peptide fragment.

From the above results, it was demonstrated that the chemically modified lysyl endopeptidase-containing composition of the present invention specifically adsorbs a peptide whose C-terminal amino acid is Lys, and it is confirmed that the composition exhibits novel affinity ligand activity. .

3-2) Fractionation by immobilized chemically modified lysyl endopeptidase-containing composition of a tryptic digest of α-neoendorphin α-neoendorphin (manufactured by Peptide Institute) 400 nmo
20 mM borate buffer pH 8.0 with 2 nmol trypsin
A solution digested in 1 ml (containing 20 mM CaCl ₂ ) at 25 ° C. for 6 hours was diluted with a starting buffer to a final concentration of 100 nmol / ml, and applied to a composition column containing immobilized chemically modified lysyl endopeptidase. The peptide was fractionated in the same manner as in 3-1) above, and the results are shown in FIG. That is, FIG. 3 is a graph showing the elution pattern when the immobilized chemically modified lysyl endopeptidase-containing composition of the present invention was subjected to a tryptic digest of α-neoendorphin. The arrow, the horizontal axis, and the vertical axis have the same meanings as in FIG. The flow-through and adsorption fractions in FIG. 3 are Tyr-Gly-Gly-Phe-Leu-Arg and Ly, respectively.
s-Tyr-Pro-Lys, which corresponded to the N-terminal peptide fragment and the C-terminal peptide fragment.

From the above results, it is shown that the chemically modified lysyl endopeptidase-containing composition of the present invention specifically adsorbs a peptide whose C-terminal amino acid is Lys, and also the modified immobilized serine protease, namely It was shown that peptide digests can be fractionated using trypsin, a protease that shows a different substrate specificity from lysyl endopeptidase.

Example 3 Separation of Peptide Mixtures Using Immobilized Chemically Modified Serine Protease-Containing Compositions Lysyl-bradykinin, α-neoendorphin and angiotensin I (all manufactured by Peptide Institute) 100 n each
Start mol buffer (50 mM acetate buffer pH 5.0) 1 m
Dissolve in l to make a peptide mixture. On the other hand, the immobilized chemically modified trypsin-containing composition column prepared in the above Example 1 was connected below the immobilized chemically modified lysyl endopeptidase-containing composition column prepared in the above Example 2 and fully equilibrated with the starting buffer. To do. Attach the peptide mixture to this duplicate column, wash thoroughly with a starting buffer,
Obtain the flow-through fraction. Then remove the ligation column and elute the peptide adsorbed on each column with 5 mM HCl (pH 2.5).
Obtain each fraction. Peptide was detected by measuring fluorescence from fluoresamine. The peptides in each fraction were identified by a peptide sequencer. As a result, angiotensin I: Asp-Arg-
Val-Tyr-Ile-His-Pro-Phe-His-Leu is adsorbed to the composition containing immobilized chemically modified lysyl endopeptidase, and α-neoendorphin: Tyr-in the eluted fraction.
Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys was confirmed, and lysylbradykinin was confirmed in the fraction adsorbed to the composition column containing immobilized chemically modified trypsin.

From the above results, separation of a peptide mixture by combining several kinds of immobilized chemically modified serine protease-containing composition columns exhibiting the novel affinity ligand activity of the present invention
It was shown that the purification could be done very easily.

Comparative Example 1 Comparison with Conventional Immobilized Chemically Modified Trypsin-Containing Composition Conventional immobilized chemically modified trypsin-containing composition prepared by the method of Ishii et al. Described above and the novel of the present invention prepared by Example 1 above. With respect to the composition containing immobilized chemically modified trypsin, the residual trypsin activity and the elution pattern of β-neoendorphin were compared and examined.

As a result, the residual trypsin activity was 0.1% of unmodified trypsin in the conventional immobilized chemically modified trypsin-containing composition, and 0.01% or more of the unmodified trypsin in the newly immobilized chemically modified trypsin-containing composition. , It was shown that it has decreased to less than 1/10.

On the other hand, the results of the elution pattern of β-neoendorphin are shown in FIG. That is, FIG. 4 is a graph comparing the performances of the conventional immobilized chemically modified trypsin-containing composition and the novel immobilized chemically modified trypsin-containing composition of the present invention. Fig. 4-1 is a graph showing the elution pattern when β-neoendorphin was added to a composition containing a novel immobilized chemically modified trypsin, and Fig. 4-2 is a graph showing the conventional immobilization of β-neoendorphin. It is a graph which shows the elution pattern when it is applied to the chemically modified trypsin-containing composition. In addition,
The arrow, the horizontal axis, and the vertical axis have the same meanings as in FIG.

The gel of the composition containing conventional and novel immobilized chemically modified trypsin was filled in each column to make a column of 6.3 ml,
Equilibrate with about 10 column volumes of starting buffer (50 mM acetate buffer pH 5.0). β-Neo Endorphin (120
(m mol / ml: manufactured by Peptide Institute) was attached to each column. Wash the column with about 20 column volumes of starting buffer.
The gel bound fraction is then eluted with 5 mM HCl (pH 2.5). Collect 3 ml fractions and perform all operations at 4 ℃.
The peptide was detected by measuring the fluorescence from fluoresamine.

In FIG. 4-1, which shows the results of the present invention, a single peak was observed only in the flow-through fraction. . This fraction was analyzed by HPLC using a reversed-phase column [Nucleosil ₅ C ₁₈ : manufactured by Nagel], and as a result, a single peak was observed at the same position as β-neo endorphin, and β-neo I knew that Endorphin had passed by.

Further, in FIG. 4-2 showing the conventional results, peaks were observed in the flow-through fraction and the adsorption fraction. Each fraction was analyzed by HPLC using a reversed phase column (Nucleosyl ₅ C ₁₈ : manufactured by Nagel). As a result, two peptides were present in the flow-through fraction, and when these were analyzed by a peptide sequencer, β- Neo endorphin (Try-Gly-Gly-Phe-Le
u-Arg-Lys-Tyr-Pro) and β-neoendorphin were found to be C-terminal peptides (Lys-Tyr-Pro) produced by cleavage by digestion with trypsin. And
The adsorbed fraction was also analyzed by reverse phase HPLC to show a single peak, which was analyzed by a peptide sequencer. As a result, β-neoendorphin was cleaved by trypsin to produce an N-terminal peptide ( Tyr-Gly-Gly-Phe-
Leu-Arg). That is, the conventional immobilized chemically modified trypsin-containing composition has a problem that the residual trypsin activity is high and cleaves the applied peptide sample, but in the novel immobilized chemically modified trypsin-containing composition of the present invention, It was shown that such problems were solved.

〔The invention's effect〕

As described in detail above, the chemically modified serine protease-containing composition of the present invention has affinity ligand activity, but since it has substantially no catalytic activity, it has a remarkable effect on separation and purification of peptides in various fields. Have.

[Brief description of drawings]

FIG. 1-1 shows the respective elution patterns when the composition containing immobilized chemically modified trypsin of the present invention was added with neurotensin, and FIG. 1-2 was shown with the tryptic digest of niurotensin. FIG. 2-1 is a graph, FIG. 2-1 shows the composition containing immobilized chemically modified lysyl endopeptidase of the present invention, and β-neoendorphin is added thereto. FIG. 3 is a graph showing each elution pattern when a digested product was applied, and FIG. 3 is an elution pattern when a trypsin digested product of α-neoendorphin was added to the composition containing immobilized chemically modified lysyl endopeptidase of the present invention. FIG. 4-1 is a graph showing that when β-neoendorphin was added to the composition containing immobilized chemically modified trypsin of the present invention, FIG. 4-2 shows the composition containing conventional immobilized chemically modified trypsin. It is a graph showing each dissolution pattern when subjected to forming thereof.

Front page continuation (72) Inventor Akira Obayashi 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Minami Shuzo Co., Ltd. (56) References Biological and Bioresearch Research, 72, (1976) P. 1443 Journal of Biochem history, 81 (1977), p. 647

Claims (3)

[Claims] 1. A composition containing a chemically modified serine protease, wherein the serine protease containing chemically modified active center serine is modified with a compound that acts only on the active center histidine of the unmodified chemically modified serine protease. Then, a chemically modified serine protease having an affinity ligand activity, but having substantially no catalytic activity, characterized by being separated by using affinity ligands having different affinities depending on whether or not the active center histidine is chemically modified. Containing composition. 2. A method for producing a composition containing a serine protease having an affinity ligand activity but substantially no catalytic activity, which is chemically modified from the active center serine, wherein the active center serine is chemically modified. It is possible to modify the crude product containing serine protease with a compound that acts only on the active center histidine of unmodified serine protease, and then to fractionate using affinity ligands having different affinities depending on the presence or absence of chemical modification of active center histidine. A method for producing a serine protease-containing composition characterized by chemically modifying active center serine. 3. A method of fractionating peptides using one or more chemically modified serine proteases,
As the serine protease, a serine protease-containing substance obtained by chemically modifying the active center serine is modified with a compound that acts only on the active center histidine of the serine protease that is not chemically modified, and then the affinity depending on the presence or absence of the chemical modification of the active center histidine. A method for fractionating peptides, which comprises using a chemically modified serine protease-containing composition having an affinity ligand activity but substantially no catalytic activity, which is obtained by fractionation using different affinity ligands.