JPH11124400A - Substrate peptide for immunoassay of serine protease derived from hepatitis c virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new substrate peptide utilizable for evaluation satisfying simplicity, rapidity and further excellent quantitativeness when evaluating the enzymic activity of a serine protease derived from hepatitis C virus. SOLUTION: This substrate peptide for the immunoassay of a serine protease derived from hepatitis C virus is a fusion protein prepared by joining the C- terminal of a maltose-binding protein derived from Escherichia coli to a peptide chain represented by the following amino acid sequence (I): Ser-Asp-Asp-Ile-Val- Cys-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-Gly-Ala-Lys-Lys-Lys-Lys-Lys and a radiolabel or a fluorescent label is added to any of the five lysine residues present at the C-terminal in the amino acid sequence (I) by chemical modification.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a substrate peptide for a serine protease derived from hepatitis C virus, and more particularly to a fusion protein type substrate peptide used in an enzyme assay. Specifically, it is a fusion protein-type substrate peptide used to measure the enzyme activity of serine protease derived from hepatitis C virus, and is useful for enzyme activity evaluation that is simple, quick, and also has excellent quantitative properties. It is suitable.

[0002]

2. Description of the Related Art In general, a method for quantitatively evaluating an enzyme activity of an enzyme protein is called an enzyme assay, which uses a certain amount of the enzyme protein and reacts the enzyme with a substrate under predetermined enzyme reaction conditions. A method is used in which the reaction is performed and the amount of the generated reaction product is quantified. At that time, the separation of the reaction product and the quantification of the reaction product from the reaction solution in a manner that does not impair the reproducibility and quantitativeness are the main factors that determine whether or not high quantitativeness is achieved. . In other words,
After the enzymatic reaction, it is necessary to easily and quickly separate the target reaction product from the reaction solution, and to remove unnecessary substances that become interference components that impair the quantification of the reaction product. The simplicity is a major factor in determining whether high quantification is achieved.

[0003] The enzyme protein to be evaluated is naturally a natural product, and the original substrate is also a natural product. However, in many cases, an artificially prepared substrate is used because of its easy availability. For example, in an enzyme assay for a protease that selects various specific amino acid sequences to cleave a peptide chain, an artificial substrate peptide that mimics the amino acid sequence of a cleavage site in a natural substrate is used. In the present invention, the amino acid sequence of a cleavage site at which the protease selectively cleaves is also known for the hepatitis C virus-derived serine protease, which is the enzyme protein of interest, and an amino acid sequence portion similar thereto is obtained by genetic recombination. Several introduced recombinant substrate proteins have been reported.

[0004] The present inventors have also developed hepatitis C virus (HC)
V) Derived serine protease; instead of p70 protein, this p70 protein itself or this p70 is added as a recombinant enzyme protein to the C-terminal of maltose binding protein derived from Escherichia coli.
Cpro-2, a region exhibiting serine protease activity in proteins
Thus, a recombinant HCV protease was prepared by linking active domains each consisting of 190 amino acids in a region referred to as a fusion protein. At the same time, the amino acid partial sequence that cleaves the above-mentioned HCV-derived serine protease Cpro-2 is changed between two proteins, specifically, E. coli-derived maltose binding protein (MBP) and pig-derived adenylate kinase enzyme protein (ADK). ) Was prepared as a recombinant substrate of a fusion protein type incorporated as a peptide linker connecting between
-9961). In addition, HCV protease
Cpro-2 selectively cleaves between Cys ... Ser and Cys ... Ala. In the above-mentioned recombinant substrate of the fusion protein type, it corresponds to the HCV protease cleavage sequence (NS5A / NS5B). An artificial peptide linker shown in FIG. 1 having the following amino acid sequence is used.

Alternatively, the present inventors have previously created a synthetic substrate peptide for HCV protease Cpro-2, and for example, the following synthetic substrate which is a peptide having an amino acid sequence corresponding to the HCV protease cleavage sequence (NS5A / NS5B). (S3): Abz-Glu- Asp-Val-Val-Glu-Cys-Ser-Met-Ser-Tyr-NH 2: also available (Abz 2-aminobenzoyl), this peptide is cleaved HCV protease Then, Cys-Ser
The following two types of peptide fragments cleaved between: Abz-Gl
u-Asp-Val-Val- Glu-Cys-OH Ser-Met-Ser-Tyr-NH 2 is obtained, such relatively small peptides found that as a substrate (Japanese Patent Application No. 9-126462 No.).

When the above-mentioned recombinant substrate of the fusion protein type is used, the generated fragment is separated from the reaction solution, and the ADK protein of the C-terminal fragment is quantified by SDS-polyacrylamide gel electrophoresis, or When a synthetic substrate peptide is used, the enzyme activity has been evaluated by separating and quantifying the generated peptide fragments by a reverse phase HPLC method or the like. In the method using these conventional substrate peptides,
Due to the restriction of the measurement time for quantifying the peptide fragment generated by the enzymatic reaction, simplicity and speed are sufficient when performing a large number of assays, for example, evaluating an inhibitor for the enzyme. It couldn't be said. Therefore, there is a demand for a new method for evaluating enzyme activity that satisfies the simplicity and speed, and that also has excellent quantitative properties, and a proposal for a new substrate peptide suitable for the method.

[0007]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems, and an object of the present invention is to provide a method for evaluating the enzymatic activity of HCV-derived serine protease Cpro-2, which is simple, fast, and easy. An object of the present invention is to provide a novel substrate peptide which can be used for a new enzyme activity evaluation method having excellent quantitative properties.

[0008]

Means for Solving the Problems The present inventors, as a substrate for the HCV-derived serine protease Cpro-2, use the above-mentioned HC
The amino acid sequence corresponding to the V protease cleavage sequence (NS5A / NS5B) is replaced with a fusion protein type recombination substrate used as an artificial peptide linker for linking the two proteins, and the C-terminal of this artificial peptide linker is By creating a fusion protein in which lysine, which is a basic amino acid, is added to the amino acid sequence in succession, in place of the adenylate kinase protein, and reacting this fusion protein with a recombinant HCV-derived serine protease, the artificial They were found to be selectively cleaved between Cys-Ser in the peptide linker. That is, by adding lysine, which is a basic amino acid, to the C-terminus in several consecutive amino acids, the artificial peptide linker is present at the N-terminus without using a bulky substance such as adenylate kinase protein. Without interacting with the maltose binding protein (MBP) moiety,
It has been found that a steric configuration that allows enzymatic cleavage by the CV-derived serine protease Cpro-2 is maintained. Furthermore, the resulting peptide fragment on the C-terminal side is characterized by the presence of the added basic amino acid lysine of several amino acids in length.
It was found that the acid-soluble fraction was selectively recovered from the reaction solution. In addition, by utilizing the basic amino acid lysine of several amino acids continuous at the C-terminal, Lys ε-
Radiolabeling with ¹⁴ C-labeled acetyl group substituted on amino group or fluorescent labeling with fluorescein isothiocyanate (FITC) bonded to Lys ε-amino group should be possible in advance. I also checked. Therefore, the peptide fragment on the C-terminal side separated and recovered by the above-mentioned simple operation can be quantified simply, quickly and with high quantification by using the radioactive label or the fluorescent label. I found that. Based on such a series of findings, the present invention has been completed.

Specifically, the hepatitis C virus-derived serine protease enzyme assay substrate peptide of the present invention comprises the following amino acid sequence (I): Ser-Asp-Asp-Ile-Val- at the C-terminal of maltose-binding protein derived from Escherichia coli. Cys-Cys-Ser-Met-Ser-Tyr-Thr-Tr
This is a fusion protein comprising peptide chains represented by p-Thr-Gly-Ala-Lys-Lys-Lys-Lys-Lys, and 5 lysine residues present at the C-terminus in the amino acid sequence (I). A radioactive label or a fluorescent label is added to any of the groups by chemical modification. In particular, the radioactive label added by the chemical modification is a substrate peptide in which the acetyl group labeled with ¹⁴ C substituted on the ε-amino group of Lys, or the fluorescent label added by the chemical modification is: Preferably, the substrate peptide is a FITC atom group bonded to the ε-amino group of Lys.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The hepatitis C virus (HC
V) A substrate peptide for a serine protease enzyme assay derived therefrom and a method for producing the same are described below. First,
This substrate peptide is obtained by linking the amino acid sequence (I) to the C-terminus of a maltose binding protein derived from Escherichia coli. This amino acid sequence (I) is the same as that of the fusion protein type already reported by the present inventors. Recombinant substrate (JP-A-9-99)
No. 61), that is, the following amino acid sequence (II): Ser-Asp as a peptide linker connecting between maltose binding protein (MBP) derived from E. coli and adenylate kinase enzyme protein (ADK) derived from pig. -Asp-Ile-Va
It has a cleavage site similar to a recombinant substrate abbreviated as MBP-NS5A / 5B-ADK incorporating l-Cys-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-Gly. That is, when the HCV-derived serine protease is allowed to act, the amino acid sequence (I)
Which is selectively cleaved between Cys-Ser in the following amino acid sequence (III): Ser-Met-Ser-Tyr-Thr-Trp-Thr-Gly-Ala-Lys-Lys-Lys-Ly
It produces a peptide fragment represented by s-Lys.

On the other hand, the most characteristic point of the substrate peptide of the present invention is that Ala-Lys-Lys-Ly existing at the C-terminal thereof.
It is part of s-Lys-Lys. Following the hydrophobic amino acid Ala, it has a long side chain and a structure in which five lysines exhibiting basicity are continuously present. In particular, since the five consecutive lysines have high hydrophilicity, the peptide chain portion of the amino acid sequence (I) causes an unnecessary interaction with the N-terminal fusion partner MBP. It plays a role in preventing a change to a structure that inhibits the serine protease Cpro-2 from approaching the enzyme active site. That is, in the recombinant substrate abbreviated as MBP-NS5A / 5B-ADK, by disposing a bulky ADK protein, the peptide chain of the amino acid sequence (II) is maintained in an elongated shape as if it were a string. However, the role is achieved by only five consecutive lysines.

The Ala-Lys-Lys-Lys-Lys-Lys portion is also used when a radioactive or fluorescent label is added to the ε-amino group of Lys by chemical modification.
Since a lysine having a long side chain is continuously present after the hydrophobic amino acid Ala, the chemical modification selectively proceeds only to a specific lysine. Therefore, the radioactive label or the fluorescent label added by the chemical modification is introduced in an equivalent amount per one molecule of the substrate peptide, and the peptide fragment to be produced using the radioactive or fluorescent label is high. It is possible to determine quantitatively. In addition, when separating the unreacted substrate peptide in the reaction solution from the peptide fragment generated by the enzymatic reaction, the basic amino acid lysine is continuously present in the generated peptide fragment. However, since the molecular weight is much smaller than that of the substrate peptide, it is selectively recovered in the acid-soluble fraction, and separation from the unreacted substrate peptide or enzyme protein can be performed by a simple, easy and quick operation. Has become. In addition, since the quantification of the generated peptide fragment portion recovered in the acid-soluble fraction uses a radioactive label or a fluorescent label, the time required for the measurement is compared to the separation and quantification by a reversed-phase HPLC method or the like. Therefore, it is extremely short, and is excellent in terms of simplicity and reproducibility.

Next, as described below, the recombinant substrate peptide of the present invention (hereinafter abbreviated as MBP-NS5A / 5B).
Is obtained by linking the amino acid sequence (I) to the C-terminus of a maltose binding protein derived from Escherichia coli.
Using a method similar to the method for producing a recombinant substrate of P-NS5A / 5B-ADK, DNA encoding the amino acid sequence (I) is used.
A fragment is synthesized, ligated to the end of a DNA encoding a maltose binding protein derived from Escherichia coli so as to be translated into a series of peptide chains, and the target fusion protein is produced in the host Escherichia coli by genetic recombination. it can.

The recombinant substrate peptide MBP-NS5A / 5B of the present invention
Recombinant Escherichia coli used for expression of the fusion protein can be obtained by the following method. Commercially available MBP expression plasmid vector (manufactured by NEW ENGLAND BIOLABS (NEB)) pMAL-c2
Was digested with restriction enzymes Xmn I and Hind III (C.-d.
l., Gene, 67 , 21-30 (1988), CV Maina et al., Gen.
e, 74 , 365-373 (1988)) and the resulting MBP
A vector fragment containing DNA (malE) encoding is purified. On the other hand, a DNA fragment encoding the peptide partial chain represented by the amino acid sequence (I) is separately prepared by a DNA synthesis method. The ends of this synthetic DNA fragment were treated with restriction enzymes Xmn I and Hind III, respectively.
It was then purified to that shown in FIG. The vector fragment containing the MBP-encoding DNA (malE) and the DNA fragment encoding the amino acid sequence (I) shown in FIG. 4 were ligated by T4 DNA ligase. The resulting plasmid vector contains a DNA encoding the MBP-NS5A / 5B fusion protein and a pMAL-c2-derived tac promoter (Pta
c), and has an ampicillin resistance gene (Amp ^r ) as a marker gene. This MBP-NS
E. coli using 5A / 5B fusion protein expression vector pMAL-5AB
Transform into JM109 strain. Ampicillin resistance by a marker gene, and screening by Western blotting using a rabbit anti-MBP antibody were performed.
A recombinant E. coli JM109 / pMAL-5AB strain having the MBP-NS5A / 5B fusion protein expression vector pMAL-5AB was obtained. This MBP-
The NS5A / 5B fusion protein expression vector pMAL-5AB was
The transformed strain introduced into the li JM109 strain was submitted to the Institute of Biotechnology, Institute of Biotechnology, Industrial Science and Technology as a strain of E. coli JM109 / pMAL-5AB designated by the inventor on September 2, 1997.
Deposited under accession number FERMP-16442 on 4th.

As the host E. coli, in addition to E. coli JM109, JM105, HB101, SCS1, BL21 (DE3),
Commercially available MBP expression plasmid vector such as BL21 (DE3) / pLys strain (manufactured by NEW ENGLAND BIOLABS (NEB)) pMAL
Using various strains that can be transformed using -MB, the MBP-
A recombinant bacterium having the NS5A / 5B fusion protein expression vector pMAL-5AB can be obtained. That is, the MBP-NS5A / 5B fusion protein expression vector pMAL-5AB is a commercially available MBP expression plasmid vector (NEW ENGLANDBIOLABS
Compared with pMAL-c2 (manufactured by (NEB)), there is only a slight difference in the portion encoding the amino acid sequence (I), and in terms of transformation efficiency, the MBP expression plasmid vector pMAL-c2
There is no substantial difference from 2, and the protein translated therefrom has only a slight difference, and does not cause a substantial difference in its productivity.

The recombinant bacterium having the expression vector pMAL-5AB was ligated with 1 L of 2 × YT (10 g / l Bactotryptone, 10 g / l
Bacto yeast extract, 5 g / l sodium chloride &
Using a medium containing 100 mg / l ampicillin), shake and culture in 12 5 L Erlenmeyer flasks (12 L in total) at 30 ° C overnight (about 14 hours). Meanwhile, during the logarithmic growth phase,
Isopropyl-1-thio-β so that the final concentration is 0.1 mM.
-D-galactoside (IPTG) was added to the MBP-NS5A / 5
B Induces expression of the fusion protein. Thereafter, the culture solution is collected, and the cells are disrupted and ultracentrifuged to obtain 600 ml of cell lysate. This soluble fraction was applied to an amylose column (5 × 10 cm) equilibrated with an amylose gel buffer (20 mM Tris-HCl, pH 7.4 / 0.2 M NaCl / 1 mM EDTA), and washed with the same buffer. Elute the adsorbed fraction with the same buffer containing 10 mM maltose. The eluted fraction containing the MBP-NS5A / 5B was subjected to ultrafiltration (YM-30 membrane, Amicon) at 3 mg / ml.
Concentrate until: If you do not sign immediately, -8
Store at 0 ° C.

As the radioactive label by chemical modification, for example, [ ¹⁴ C] acetic anhydride is used to add ¹⁴ C on the ε-amino group of Lys.
By versatile means such as replacing the acetyl group labeled with
^It is good to perform N-acylation with an acyl group labeled with ¹⁴ C,
In enzymatic reactions, it does not cause steric hindrance ¹⁴
Acetyl groups labeled with C are more preferred. As the fluorescent label by chemical modification, various fluorescent labels that are widely used can be used.
It is more preferable to use a C atom group. That is, the radiolabeling with an acyl group labeled with ¹⁴ C or the fluorescent labeling with a FITC atom group does not hinder the quantification even when various proteins and peptides are mixed. It will be preferable. The specific method of radiolabeling is described below.

The concentrated MBP-NS5A / 5B fusion protein solution was equilibrated with a borate buffer (10 mM Na ₂ B ₄ O ₇ , pH 9.0 / 1 mM DTT) on Sephadex G-25 medium (2.2 × 26 cm
) And perform solvent exchange. To 24 ml of the MBP-NS5A / 5B fusion protein solution replaced with the borate buffer, add 2 ml
Was dissolved in dioxane [1- ¹⁴ C] acetic anhydride (18.5 MBq,
3.7 to 4.59 Gbq / mmol, Amersham)
After dissolving in 1 ml of dioxane and adding to the protein solution, wash the ampoule with 1 ml of dioxane and add)
Stir in ice for 30 minutes to acetylate the ε-amino group of the C-terminal Lys. After the reaction, a TSC buffer containing 1 mM DTT (50
(Tris-HCl, pH 8.5 / 30 mM NaCl / 5 mM CaCl ₂ ) to completely remove the reaction by-product [ ¹⁴ C] acetic acid. In this way, labeled MBP-NS with a specific activity of 1.1 × 10 ⁶ dpm / mg
36 mg of 5A / 5B is obtained. DTT to a final concentration of 25 mM
And adjust the protein concentration to 1 mg / ml, and store at -80 ° C. The present invention will be described in more detail with reference to specific examples. The MBP-p7 used in the following specific examples
0 Fusion protein expression vector pMAL-CP and conventional MBP-NS5A / 5
Figure 2 shows a schematic diagram of the B-ADK fusion protein expression vector pMAL-AK2.
Is shown for reference.

[0019]

EXAMPLE The substrate peptide for the enzyme assay for serine protease derived from hepatitis C virus of the present invention was actually synthesized with HCV protease to form Cys- in the amino acid sequence (I).
The following specific examples show that quantification of the C-terminal peptide fragment generated at the time of cleavage between Sers can be performed easily, easily, and with excellent quantification. The natural HCV protease Cpro-2 is produced in the host cell as the polypeptide p70 and is difficult to obtain in large quantities.
A recombinant hepatitis C virus protease having a protease activity equivalent to that of Cpro-2 was produced by a genetic engineering technique using a transformed microorganism and used.

Enzyme activity test method In this example, recombinant HCV was used as the HCV protease.
Using a protease, it was evaluated that the recombinant protein of the present invention could be used as a substrate. [Preparation of Recombinant HCV Protease of MBP-p70 Fusion Protein Type] Recombinant HCV protease is prepared by following the procedure described below.
A circular plasmid vector containing A is constructed, a transformed microorganism having the ability to produce the recombinant HCV protease is prepared by introducing the transformed circular plasmid vector, and the transformed microorganism is cultured. Then, the recombinant HCV protease was isolated and collected from the culture to prepare it (see Japanese Patent Application Laid-Open No. 9-9961). In addition,
The cDNA encoding a HCV-derived protein such as HCV protease is composed of the HCV viral genome (+ chain RN).
The nucleic acid sequence and the corresponding amino acid sequence obtained by reverse transcription from A) have already been reported in the literature (A. Tak
amizawa et al., J. Virol., 65 , 1105-1113 (1991), N.
Kato et al., Proc. Natl. Acad. Sci. USA, 87 , 9524.
-9528 (1990)). In this example, the HCV-J genomic nucleic acid sequence (N. Kato et al., Proc. Natl. Acad.
Sci. USA, 87 , 9524-9528 (1990), etc.).

The recombinant HCV protease is a maltose-binding protein (MBP; maltose-binding protein) derived from Escherichia coli.
A fusion protein in which HCV protease was linked to the C-terminus of protein (protein) was used (see Japanese Patent Application Laid-Open No. 9-9961). A commercially available MBP expression plasmid vector (NEW ENGLAND BIOLABS (NEB)) pMAL-c2 was replaced with a restriction enzyme.
Cleavage with EcoR I and Hind III (C.-d.Guan et al., Gene,
67 , 21-30 (1988), CVMaina et al., Gene, 74 , 365.
-373 (1988)), and the resulting vector fragment containing DNA (malE) encoding MBP was purified. And the vector fragment, said p70; a DNA fragment encoding the Ala ¹⁰²⁷ ~ Thr ¹⁶⁵⁸ connected by T4DNA ligase to obtain MBP-p70 expression vector pMAL-CP. The obtained plasmid vector was constructed by adding a DNA encoding the MBP-p70 fusion protein to a tac promoter (P
tac) and an ampicillin resistance gene (Amp ^r ) as a marker gene. This MBP-
Using the p70 fusion protein expression vector pMAL-CP, E. coli HB
101 strains were transformed. Ampicillin resistance by a marker gene and screening by Western blotting using a rabbit anti-MBP antibody were performed to obtain a recombinant bacterium having the expression vector pMAL-CP.

Culture production of fusion protein by recombinant bacteria and isolation of active recombinant HCV protease from culture
Purification was performed in the following procedure. 100 mg / l of recombinant bacteria
And shake culture in a 5 L Erlenmeyer flask at 30 ° C. overnight (about 14 hours) using 500 ml of 2 × YT medium to which ampicillin was added. During this period, during the logarithmic growth phase, the medium was treated with isopropyl-1-methyl to induce the expression of the MBP-p70 fusion protein.
Thio-β-D-galactoside (IPTG) is added to a final concentration of 0.1 mM. Subsequently, shaking culture was performed at 30 ° C. for 14 hours, and the obtained culture solution was centrifuged to collect bacteria. After collecting the cells, the cells were disrupted by ultrasonic waves, and then the insoluble fraction was separated by centrifugation to obtain a supernatant containing a soluble protein.

The above supernatant collected from 2 L of the culture solution 137.
5 ml of amylose gel buffer (20 mM Tris-HCl, pH 7.
4 / 0.2 M NaCl / 1 mM EDTA), equilibrated to an amylose column (32 × 63 mm; NEW ENGLAND BIOLABS), washed with the same buffer, and adsorbed on the column.
The P-p70 fusion protein was eluted with the same buffer supplemented with 10 mM maltose. The adsorbed fraction was concentrated by ultrafiltration (YM-30 membrane, manufactured by Amicon). This concentrated solution is subjected to gel filtration buffer (0.2 M NaCl / 0.1 M sodium phosphate, pH 7.2)
S-300 HR column (Phar
macia) and gel filtration column chromatography. Flow rate 400 ml / h, fractions 20 ml each
And SDS-PAGE was performed, and fractions containing the target MBP-p70 fusion protein were pooled. This gel filtration was performed
The fraction containing the MBP-p70 fusion protein is again concentrated on an ultrafiltration membrane. Add this to 20 mM sodium phosphate buffer, pH
The solution was applied to a Sephadex G-25 column equilibrated with 7.2 (hereinafter referred to as buffer B), and the solvent was replaced with buffer B to obtain an MBP-p70 fusion protein solution. Finally, glycerol was added to the purified MBP-p70 fusion protein solution to a final concentration of 50%, and the protein concentration was 1 mg / m2.
After adjusting to l, it was stored at -80 ° C. Next, the recombinant substrate peptide MBP-NS5A / 5B of the present invention was prepared by the following procedure.

[Preparation of MBP-NS5A / 5B fusion protein]
Recombinant E. coli used for expression of the recombinant substrate peptide MBP-NS5A / 5B fusion protein was obtained by the following method. A commercially available MBP expression plasmid vector (NEW ENGLAND BIOLABS
(NEB)) pMAL-c2 was digested with restriction enzymes Xmn I and Hind III (C.-d. Guan et al., Gene, 67 , 21-30 (1988), CV
(See Maina et al., Gene, 74 , 365-373 (1988), etc.)
The resulting vector fragment containing DNA (malE) encoding MBP was purified. On the other hand, a DNA fragment encoding the peptide partial chain represented by the amino acid sequence (I) was
It was produced by the R method. That is, amplification was performed using the MBP-NS5A / 5B-ADK expression vector pMAL-AK2 (see JP-A-9-9961 and the like) as a template and the following synthetic primers.

[0025]

Embedded image

The ends of the DNA fragment were treated with the restriction enzymes Xmn I and Hind III, respectively, at the restriction enzyme cleavage sites present in the primers, and then purified. DNA encoding the MBP (malE)
And a DNA fragment encoding this amino acid sequence (I) were ligated with T4 DNA ligase, and incorporated into the downstream of DNA (malE) encoding MBP as shown in FIG. The obtained plasmid vector was constructed by introducing the DNA encoding the MBP-NS5A / 5B fusion protein into pMAL.
-c2 is located downstream of the tac promoter (Ptac) and is used as a marker gene for the ampicillin resistance gene.
(Amp ^r ). Using the MBP-NS5A / 5B fusion protein expression vector pMAL-5AB, E. coli JM109 strain was transformed. Screening was performed by ampicillin resistance using a marker gene and Western blotting using a rabbit anti-MBP antibody, and a recombinant bacterium E. coli JM1 having the MBP-NS5A / 5B fusion protein expression vector pMAL-5AB was screened.
09 / pMAL-5AB was obtained. This recombinant bacterium E. coli JM109 / pMA
L-5AB has been deposited with the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-16442. The nucleotide sequence of the PCR amplification region in the expression vector was confirmed by the Sanger method using a commercially available sequencing kit; BcaBEST DNA Sequencing Kit (Takara Shuzo).

The recombinant bacterium having the expression vector pMAL-5AB was mixed with 1 L of 2 × YT (containing 100 mg / l ampicillin).
Using a medium containing
L) at 30 ° C. overnight (about 14 hours) with shaking culture. Meanwhile, during the logarithmic growth phase, IPTG was added to a final concentration of 0.1 mM to induce the expression of the MBP-NS5A / 5B fusion protein. Thereafter, the culture solution was collected, and the cells were disrupted and ultracentrifuged to obtain 600 ml of cell lysate. This soluble fraction was added to an amylose gel buffer (20 mM Tris-HCl, pH 7.4 / 0.2 M NaCl / 1 mM
Amylose column (5 x 10 cm) equilibrated with EDTA
After washing with the same buffer, the adsorbed fraction was eluted with the same buffer containing 10 mM maltose. This MBP-NS5A
The eluted fraction containing / 5B is ultrafiltered (YM-30 membrane, Am
icon) to 3 mg / ml.

[Radiolabeling of MBP-NS5A / 5B fusion protein] The concentrated MBP-NS5A / 5B fusion protein solution was treated with a borate buffer (10 mM Na ₂ B ₄ O ₇ , pH 9.0 / 1 mM DTT). The solution was applied to an equilibrated Sephadex G-25 medium (2.2 × 26 cm 2), and solvent exchange was performed. MB replaced with this borate buffer
P-NS5A / 5B fusion protein solution 24 ml, was dissolved in a 2 ml dioxane [1- ¹⁴ C] acetic anhydride (18.5 Mbq, 3.7 ~4.59 Gbq
/ mmol, manufactured by Amersham) (dissolve in 1 ml of dioxane first, add to the protein solution, wash the ampoule with 1 ml of dioxane and add), stir in ice for 30 minutes, The ε-amino group of Lys was acetylated. After the reaction, a TSC buffer containing 1 mM DTT (50 mM Tris-HCl,
The mixture was dialyzed against pH 8.5 / 30 mM NaCl / 5 mM CaCl ₂ ) to completely remove the reaction by-product [ ¹⁴ C] acetic acid. using this method,
36 mg of labeled MBP-NS5A / 5B having a specific activity of 1.1 × 10 ⁶ dpm / mg was obtained. DTT was added to a final concentration of 25 mM, the protein concentration was adjusted to 1 mg / ml, and the mixture was stored at -80 ° C.

[Labeling of MBP-NS5A / 5B fusion protein with FITC] 1.5 mg of FITC was added to 24 ml of the MBP-NS5A / 5B fusion protein solution solvent-exchanged with the above borate buffer, and the mixture was added at 25 ° C.
The mixture was stirred under light shielding. After 3 hours, the reaction solution was cooled to 4 ° C., and dialyzed against a TSC buffer containing 1 mM DTT to remove FITC non-specifically adsorbed to the fusion protein. Add DTT to a final concentration of 25 mM and reduce the protein concentration to 1
After adjusting to mg / ml, the mixture was stored at -80 ° C with shielding from light.

FIG. 3 shows the results of analysis by SDS-PAGE in each process of production, recovery from culture, and purification of the MBP-NS5A / 5B fusion protein by the recombinant bacterium E. coli JM109 / pMAL-5AB. The induction of IPTG caused the expression of the MBP-NS5A / 5B fusion protein, which was recovered in the soluble fraction when the cells were disrupted. The MBP-NS5A / 5B fusion protein can be purified and recovered in the course of separation using an amylose column. In FIG. 3, lane 1 is a molecular weight marker, lane 2 is whole cell protein before IPTG induction, lane 3 is whole cell protein at the end of culture after IPTG induction, and lane 4 is an ultrasonic wave of cells. Supernatant after crushing and ultracentrifugation, lane 5
Is the precipitate after sonication and ultracentrifugation of the cells, lane 6
Unadsorbed fraction of the first amylose column, lanes 7 to 9
Is the first amylose column adsorption fraction, lane 10
Is the fraction not adsorbed on the second amylose column, lane 1
1 shows each analysis result of the second amylose column adsorbed fraction.

The host E. coli is E. coli JM described above.
In addition to 109, JM105, HB101, SCS1, BL21 (DE
3) Commercially available MBP such as strain, BL21 (DE3) / pLys strain
Expression plasmid vector (NEW ENGLAND BIOLABS (NEB)
Recombinant bacteria having the MBP-NS5A / 5B fusion protein expression vector pMAL-5AB can be obtained using various strains that can be transformed using pMAL-c2. Was used to produce the MBP-NS5A / 5B fusion protein. FIG. 5 shows a comparison of the results of SDS-PAGE analysis of all the cell proteins collected from the cultured cells of these recombinant cells. In FIG. 5, lane 1 is a molecular weight marker, lane 2 is a whole cell protein after IPM induction of JM109 / pMAL-5AB, and lane 3 is a whole cell protein before IPTG induction of JM105 / pMAL-5AB. 4 is the whole cell protein after IPM induction of JM105 / pMAL-5AB, lane 5 is the whole cell protein before IPB induction of HB101 / pMAL-5AB, and lane 6 is the IPB of HB101 / pMAL-5AB.
Total cell protein after TG induction, lane 7 shows SCS1 / pMAL-5AB
Lane 8 shows whole cell protein before IPTG induction of SCS1 / pMAL-
Lane 9 shows whole cell protein after IPTG induction of 5AB, BL21 (D
E3) / pMAL-5AB whole cell protein before IPTG induction, lane 10
Is the whole cell protein after IPTG induction of BL21 (DE3) / pMAL-5AB,
Lane 11 is the whole cell protein before IPTG induction of BL21 (DE3) / pLys, pMAL-5AB. Lane 12 is BL21 (DE3) / pLys, pMAL
5 shows the results of analysis of all bacterial proteins after IPTG induction of -5AB. FIG. 5 shows the expression level (relative value) of the MBP-NS5A / 5B fusion protein in each recombinant strain when IPTG induction was performed.
Below.

Radioactive labels by chemical modification include:
[ ¹⁴ C] The acetyl group labeled with ¹⁴ C was substituted on the ε-amino group of Lys using acetic anhydride. In addition, the most commonly used FIT as a fluorescent label by chemical modification is FIT.
The C group was used.

[In vitro HC using recombinant substrate protein]
V Protease Activity Assay] In vitro HCV protease activity was measured using the HCV protease fusion protein MBP-p70 and the recombinant substrate protein MBP-NS5A / 5B prepared by the above method. One example of an assay procedure is described below.

As the enzyme solution, the above-mentioned stored enzyme solution having a protein concentration of 1 mg / ml was diluted 40-fold with a diluting buffer (10 mM sodium phosphate, pH 7.2 / 50% glycerol). . Once a predetermined amount of the test substance is dissolved in DMSO,
A sample solution was used. 2 μl of this sample solution contained 66 μl of TSC buffer without DTT and TS containing 50% glycerol.
C buffer 10 μl, enzyme dilution 10 μl, 0.25 mM NS4A
2 μl of the peptide was added and mixed. After preincubation at room temperature for 10 minutes, radiolabeled MBP-NS5A / 5B (1.
The reaction was started by adding 10 μl of 2 mg / ml), and the reaction was carried out at 37 ° C. for 1 hour. The NS4A peptide is a peptide derived from HCV and has the following amino acid sequence. Natural H
Forming a protein-protein bond with CV protease p70,
It has been found that it has a function of keeping V protease p70 as an active enzyme, and the same function is exerted on the HCV protease fusion protein MBP-p70 used in this example. In this example, the NS4A peptide used was artificially prepared separately.

Amino acid sequence of NS4A peptide Leu-Thr-Thr-Gly-Ser-Val-Val-Ile-Val-Gly-Arg-Ile-Il
e-Leu-Ser-Gly-Arg-Pro-Ala-Val-Val-Pro-Asp Then, the reaction was stopped by adding 25 μl of 10% trichloroacetic acid (TCA), and the HCV protease fusion protein was centrifuged. MBP-p70, unreacted recombinant substrate protein MBP-
NS5A / 5B and C-terminally truncated recombinant substrate protein
MBP-NS5A / 5B was removed as an insoluble fraction, and only the C-terminal peptide fragment was recovered in the acid-soluble fraction. A part of the acid-soluble fraction was radiolabeled, and the radioactivity was measured using a liquid scintillation counter (TRI-CARB,
PACKARD). Similarly, for those labeled with FITC, fluorescence was measured with a general-purpose fluorometer.

As an example, FIG. 6 shows MBP-NS labeled with ¹⁴ C.
The time course of the enzymatic activity of HCV protease fusion protein (MBP-p70) to which NS4A peptide was added using 5A / 5B as a substrate is shown. It was confirmed that the radioactivity of the TCA-soluble fraction increased with the reaction time. That is, MBP-NS5A / 5
It was confirmed that the NS5A / 5B site of the B substrate was cleaved, and the C-terminal peptide containing lysine labeled with ¹⁴ C was released. In FIG. 6, at 30 minutes after the start of the reaction, the HCV protease fusion protein of the substrate MBP-NS5A / 5B was used.
Regarding the cutting speed by MBP-p70, please refer to MBP-NS5A / 5B-ADK
And the recombinant substrate protein MBP-NS5A / 5B,
No difference in the speed was found within experimental error. On the other hand, for quantitativeness, radiolabeling or FI
Since the fluorescent label of TC was used, the improvement was remarkable. The synthetic substrate (S3): Abz-Glu- Asp-Val-Val-Glu-Cys-Ser-Met-Ser-Tyr-NH 2 (Abz: 2- aminobenzoyl) compared to the quantitative property when using But it was nothing less than comparable. In particular, considering the reproducibility in repeated measurement, the reverse phase HP
It was confirmed that the assay showed higher quantification than the assay system using a synthetic substrate (S3) analyzed by LC.

Furthermore, the presence of the remaining C-terminal peptide fragment was confirmed by adding an acidic solution again to the protein component removed as the insoluble fraction. It was found that there was no residual amount that could be measured. In this respect, it was confirmed that the reproducibility and the quantitativeness were excellent. In particular, when such an enzyme assay system is applied to the evaluation of an enzyme activity inhibitor of the HCV-derived serine protease, the lower limit of quantification does not become a major problem, and reproducibility is most important. The high reproducibility, simplicity and speed obtained by using the radioactively or fluorescently labeled recombinant substrate protein MBP-NS5A / 5B will make it more effective than other methods. .

[0038]

EFFECT OF THE INVENTION The use of the radioactive- or fluorescent-labeled recombinant substrate protein of the present invention makes the operation of the HCV-derived serine protease Cpro-2 easier, faster and more convenient in evaluating the enzymatic activity. Enzyme activity evaluation which also has a quantitative property can be performed. In particular, when the method for evaluating the enzyme activity of HCV-derived serine protease Cpro-2 using this recombinant substrate protein is used to evaluate an inhibitor of the enzyme activity, the high reproducibility and rapidity of a variety of samples It is possible to carry out the evaluation of in a short time while suppressing systematic measurement errors.

[0039]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 87 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATC GAG GGA AGG ATT TCA GAT GAT ATC GTT TGT TGT TCT ATG TCT TAC 48 Ile Glu Gly Arg Ile Ser Asp Asp Ile Val Cys Ser Met Ser Tyr 1 5 10 15 ACT TGG ACT GGT GCC AAA AAG AAG AAG AAA TAA TAA GCT 87 Thr Trp Thr Gly Ala Lys Lys Lys Lys Lys 20 25

SEQ ID NO: 2 Sequence length: 21 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Asp Asp Ile Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala 5 10 15 Lys Lys Lys Lys Lys 20

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of a recombinant substrate MBP-NS5A / 5B-ADK, an amino acid sequence of an artificial peptide linker corresponding to an NS5A / NS5B cleavage site used therein, and a corresponding DNA base sequence.

FIG. 2 is a diagram outlining a method for producing a recombinant substrate of an HCV protease fusion protein MBP-p70 and a conventional MBP-NS5A / 5B-ADK using transformants.

[Fig. 3] Recombinant substrate protein MBP-NS5A using transformed bacteria
SDS-, which indicates the purity of / 5B production and its purification process
The photograph which shows the analysis result by PAGE electrophoresis.

FIG. 4 shows the amino acid sequence of the C-terminal part of the recombinant substrate protein MBP-NS5A / 5B and the corresponding DNA base sequence (A), and the expression vector pMAL-5AB for the recombinant substrate protein MBP-NS5A / 5B.
FIG.

FIG. 5 is a photograph showing the results of analysis by SDS-PAGE electrophoresis comparing the productivity of the recombinant substrate protein MBP-NS5A / 5B using various host Escherichia coli cells transformed with the expression vector pMAL-5AB.

FIG. 6: HC using radiolabeled MBP-NS5A / 5B as substrate
The figure which shows a time-dependent change of V protease activity.

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[Procedure amendment]

[Submission date] October 23, 1997

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Figure 3

[Correction method] Change

[Correction contents]

FIG. 3

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] Fig. 5

[Correction method] Change

[Correction contents]

FIG. 5

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12N 1/21 C12N 9/50 9/50 C12P 21/02 C C12P 21/02 C12N 15/00 ZNAA (C12N 1/21 (C12R 1:91) (C12N 9/50 C12R 1:92) (C12P 21/02 C12R 1:91)

Claims (3)

[Claims] The present invention relates to a maltose binding protein derived from Escherichia coli.
The following amino acid sequence (I) at the end: Ser-Asp-Asp-Ile-Val-Cys-Cys-Ser-Met-Ser-Tyr-Thr-Tr
This is a fusion protein comprising peptide chains represented by p-Thr-Gly-Ala-Lys-Lys-Lys-Lys-Lys, and 5 lysine residues present at the C-terminus in the amino acid sequence (I). A radioactive or fluorescent label attached to any of the groups by chemical modification.
Substrate peptide for serine protease enzyme assay from hepatitis B virus. 2. The substrate peptide according to claim 1, wherein the radioactive label added by the chemical modification is an acetyl group labeled with ¹⁴ C substituted on the ε-amino group of Lys. 3. The fluorescent label added by the chemical modification is a fluorescein isothiocyanate atomic group bonded to the ε-amino group of Lys.
The substrate peptide according to any one of the preceding claims.