KR100473443B1 - Process for production of protein - Google Patents

Abstract

The present invention comprises the steps of (1) transforming a host cell with a vector containing a gene encoding a fusion protein comprising a polypeptide of interest and a protective polypeptide; (2) culturing the transgenic host cell to express the gene to produce a fusion protein; And (3) cleaving the polypeptide of interest from the fusion protein with a host cell-specific protease.

According to the present invention, large amounts of the desired polypeptide can be produced at low cost. In particular, according to the present invention, a large amount of Staphylococcus aureus V8 protease is dependent on the method of genetic recombination. It can be produced efficiently and at low cost using a safe host such as E. coli.

Description

Process for production of protein

The present invention relates to a method for producing a desired polypeptide using genetic recombination techniques. Preferably the polypeptide of interest is an physiologically active polypeptide, for example an enzyme such as a protease. For example, the present invention provides an S. A method for producing derivatives of the Aureus V8 protease is provided.

As a more specific example, the present invention utilizes an E. coli expression system. Active V8 protease derivatives by expressing the insoluble fusion protein of the Aureus-derived V8 protease derivative, cleaving the V8 protease derivative from the fusion protein with an E. coli native ompT protease in the presence of a denaturing agent, and if necessary re-overlapping the V8 protease Provide a way to produce.

s. Aureus V8 protease is one of the proteases secreted into the culture medium by the Staphylococcus aureus V8 strain. This enzyme is S. It was isolated and purified in 1972 as one of the serine proteases secreted into the culture medium of the Aureus V8 strain and specifically cleaving the C-terminus of glutamic acid and aspartic acid. Jean Houmard and Gabriel R. Drapeu (1972), Proc Natl. Acad. Sci. USA, 69, 3506-3509. The DNA nucleotide sequence of this enzyme was determined in 1987. Cynthia Carmona and Gregory L. Gray (1987), Nucleic Acid Res. 15, 6757].

The enzyme is thought to be expressed as a precursor with 336 amino acid residues and secreted as a mature protein by deletion of the prepro sequence of 68 amino acid residues from the N-terminus of the precursor. It is also known that the enzyme has a repeating sequence of proline-aspartic acid-asparagine (amino acids 221 to 256) in the C-terminal region. It is not clear whether such a repeat sequence is essential for enzyme activity and Gray et al. Believe that the repeat sequence will function when present as an inactive enzyme prior to secretion.

Although the action of this enzyme has not been fully analyzed, it is widely used to measure the amino acid sequence of proteins because the enzyme specifically cleaves the C-terminus of glutamic acid and aspartic acid. In addition, since the enzyme acts on the substrate even in the presence of urea (about 2M concentration), it is possible to liberate the desired peptide from its fusion protein after solubilization using urea of a large amount of insoluble fusion protein expressed intracellularly according to genetic recombination techniques. Used.

The present inventors have successfully produced the human calcitonin by the genetic recombination technique using the above-mentioned method successfully (Japanese Unexamined Patent Publication (Kokai) No. 5-328992, EP 528686). Also, S. Aureus V8 protease was used to cleave human glucagon from fusion proteins expressed in the E. coli expression system (Kazumasa Yoshikawa et al., (1992), Journal of Protein Chemistry, 11, 517-525).

As noted above, this enzyme has been widely used in the research and production of peptides by genetic recombination. However, the enzyme s. As it is purified from the culture medium of Aureus V8, (1) the enzyme is contaminated with traces of other proteins, and (2) Arureus V8 is a pathogenic strain, and (3) there is a problem in that the product is expensive.

Therefore, it is an object of the present invention to provide S. Mass production of the desired polypeptide, such as the Aureus V8 protease. s. The production of highly purified target polypeptides, such as the Aureus V8 protease, is of great benefit for scientific research and industry and requires a method of industrial production of large amounts of polypeptide.

Therefore, the present invention

(1) transforming a host cell with an expression vector comprising a gene encoding a fusion protein comprising a polypeptide of interest and a protective polypeptide;

(2) culturing the host cell transformed to express the gene to produce a fusion protein; and

(3) A method for producing a target polypeptide, comprising cutting a target polypeptide from a fusion protein with a host cell-specific protease.

According to a preferred aspect of the present invention,

(1) a fusion protein comprising an E. coli host cell comprising at least one protective polypeptide, a desired polypeptide and a linker peptide, wherein the protective polypeptide is a polypeptide derived from E. coli β-galactosidase And / or a polypeptide derived from an aminoglycoside 3'-phosphotransferase of transposon 903 origin, the polypeptide of interest is a derivative of the Staphylococcus aureus V8 protease, between the protective polypeptide and the desired polypeptide. Transforming the linker peptide with an expression vector comprising the gene encoding the host cell-specific protease specifically recognized);

(2) expressing said gene in an E. coli host cell to produce a derivative of Staphylococcus aureus V8 protease as an inactive fusion protein;

(3) crushing the cells to separate the fusion protein and obtaining a fraction containing this cell-specific protease, the E. coli ompT protease and the fusion protein;

(4) solubilizing the fusion protein with a denaturing agent and

(5) There is provided a method for producing a target polypeptide comprising the steps of obtaining a target polypeptide from a fusion protein by cutting the linker peptide with the protease by reducing the denaturant concentration to the level at which the activity of the E. coli ompT protease is shown.

In order to carry out the invention, genes encoding the polypeptide of interest, eg S. a. Genes encoding the Aureus V8 protease are described, for example, in S. aureus. The genes thus obtained are isolated or synthesized from Aureus V8 and introduced into safe host cells, such as E. coli cells, and then the desired polypeptides, such as enzymes, are produced at low cost.

In general, in order to produce the desired polypeptide or protein by genetic engineering, it is preferable to form the desired polypeptide or protein as a fusion protein and accumulate the polypeptide or protein in the cell as an insoluble inclusion body to produce the resulting polypeptide or protein. This prevents adverse effects on the growth and survival of the host cell and on the expression of the desired polypeptide or protein.

However, in some cases according to conventional methods, fusion proteins comprising the desired polypeptide or protein do not form inclusion bodies. In such cases, according to the present invention both the C-terminus and N-terminus of the polypeptide or protein of interest are linked to the protective peptide via a linker peptide to form an insoluble fusion protein to form inclusion bodies.

According to an aspect of the present invention, the fusion protein comprising the desired polypeptide or protein accumulates intracellularly as inclusion bodies. Although the host cell-specific protease does not act on the inclusion body, after the inclusion body is isolated and the fusion protein is dissolved, such protease accompanying the inclusion body acts on the fusion protein to cleave the fusion protein to release the desired polypeptide or protein. May result. In this way, according to the present invention, high purity target polypeptides or proteins can be efficiently produced using genetic engineering.

As an example of the production of a polypeptide or protein of interest according to the invention, an S. coli expression system is used. A method for the large scale production of high purity of the Aureus V8 protease is described in detail.

First of all, the inventors found that S. using E. coli expression system. For mass production of the Aureus V8 protease, it was considered desirable to express the protease as an enzymatically inactive fusion protein because the protease hydrolyzes the E. coli protein when the protease is expressed directly in the host cell. This leads to termination of growth of the host cell and a large amount of S. a. It was thought that Aureus V8 protease could not be obtained.

Thus, the inventors expressed the protein of interest as an enzymatic inactive fusion protein and cleaved the enzymatically active Staphylococcus aureus V8 protease moiety from the fusion protein using another protease to re-overlap the cleaved V8 protease. Next, it was planned to purify the active Staphylococcus aureus V8 protease.

The inventors also found that E. coli as a protease for fusion protein cleavage. The use of ompT protease, which is thought to be present in the outer membrane of E. coli cells, was considered, because the addition of other enzymes was thought to increase the cost of production.

E. coli cell-specific ompT protease use is (1) simple to produce; (2) the need for addition of additional enzymes into the reaction system results in low production costs; (3) incorporation of the contaminated protein into the product of a V8 protease preparation (for commercially available V8 proteases, S. aureus-derived proteins), since there is no need to use separately produced V8 proteases by other hosts. This has the advantage that it can be avoided.

this. The coli ompT protease is present in the E. coli outer membrane fraction and selectively cleaves the bond between two basic amino acids. Keijiro Sugimura and Tatsuro Nishihara (1998), J. Bacteriol. 170, 5625-5632. Sugimura et al. Purified the ompT protease and cleaved various peptides at 25 ° C. for 30 minutes. Reported cutting. However, they did not mention whether the ompT protease was active in the presence of urea (more than 2M of urea). ompT protein occurs in the outer membrane, so S. The aureus V8 protease is produced as an E. coli intracellular inclusion, the E. coli cells are crushed, and then the centrifuge is separated to separate the insoluble fraction and the ompT protease is co-precipitated with the precipitate fraction.

Urea was added to the precipitate fraction thus obtained, thereby obtaining S. Solubilize the fusion protein containing the Aureus V8 protein. We believe that if ompT protease activity is maintained under these conditions, the ompT protease is derived from the fusion protein. It can be used to cleave the Aureus V8 protease and by a simple method by re-overlapping the cleaved protease to regenerate the active enzyme. It was thought that Aureus V8 protease could be produced.

In the following, the present invention is described in detail. s. Based on the nucleotide sequence of the Aureus V8 protease gene, the signal sequence (presequence) required for secretion and the pro-featured pro sequence are present at the N-terminus of the mature protein and the aforementioned repeat sequence is at the C-terminus of the mature protein. It is reported to exist. Thus, gene I, which encodes a mature protein from the N terminus to the C terminus and a gene II lacking a repetitive sequence of unknown function, is prepared. Although it is not clear whether a repetitive sequence of unknown function is essential for enzymatic activity, the inventors have found that if this repetitive sequence is not essential, the present invention results in an increase in the number of molecules of the expressed protein per cell by deleting the repetitive sequence and decreasing its molecular weight. It was thought that the amount of protein could be increased.

Thus, chromosomal DNA preparations are isolated from Staphylococcus aureus V8 (ATCC 27733) and two V8 protease derivative genes I and II are prepared by PCR. To express these derivatives, the plasmids pV8PRT (+) and pV8PRT (-), in which E. coli β-galactosidase derivatives are used as protective peptides in fusion proteins, are constructed. In these plasmids, genes encoding E. coli β-galactosidase derivatives and genes encoding V8 protease derivatives (I or II) are recognized as ompT proteases to express fusion proteins under the control of the lactose promoter. To a gene encoding a linker peptide containing arginine-arginine.

For fusion proteins designed as such, the V8 protease derivative is expressed as an insoluble fusion protein, solubilizing the fusion protein using urea, and the urea so that the E. coli β-galactosidase derivative protein and the V8 protease derivative protein are separated. It is thought that it is possible to cleave the fusion protein with ompT protease by decreasing the concentration.

A plasmid designed as above was constructed to induce expression of the fusion protein in E. coli host cells using isopropyl-β-D-thio-galactopyranoside (IPTG). As a result, the two fusion proteins were not insoluble, and enzyme activity was detected in the supernatant after crushing the cells.

On the other hand, the growth of cells significantly decreased after induction with IPTG. Analysis by SDS polyacrylamide gel electrophoresis showed that the intracellular protein was degraded by the enzymatic activity of the expressed V8 protease derivative. Thus, in a method of expressing a fusion protein comprising an E. coli β-galactosidase derivative and a V8 protease derivative, (1) the expressed enzyme has enzymatic activity and inhibits the growth of a host cell; (2) It was evident that the method was not suitable for the production of V8 protease derivatives because the amount of expressed protein was extremely low.

However, from the foregoing results, (1) the N-terminal pro-sequence probably does not involve re-overlap of the V8 protease; (2) Since the V8-protease derivative II having no C-terminal repeat sequence is active, it is believed that the repeat sequence is not essential for V8 protease activity.

The V8 protease is then expressed as an insoluble fusion protein in an E. coli expression system, solubilizing the fusion protein with urea, using the protease in the presence of urea to release the V8 protease from the fusion protein and re-overlap the released V8 protease. In order to produce an active V8 protease, we set out to experiment based on the following assumptions.

(1) When the aforementioned E. coli β-galactosidase was fused to the N-terminus of the V8 protease, the resulting fusion protein was not insoluble. Thus, we planned to add additional protective peptides to the fusion proteins described above to form insoluble fusion proteins, using aminoglycoside 3′-phosphotransferase proteins derived from the kanamycin resistance gene of transposon 903. Attempts are made in Nucleic Acids Res. (1998) 16, 358. In other words, the present inventors have described aminoglycosides via linker peptides to promote insolubilization of the fusion protein to form inclusion bodies at the C-terminus of the fusion protein comprising an E. coli β-galactosidase derivative and a V8 protease. Consider adding a portion of the 3-phosphor transferase protein.

In addition, (2) the inventors have found that if the aforementioned R6 linker is located at the N- and C-terminus of the V8 protease, the V8 protease will be released from the fusion protein by cleaving the fusion protein with an ompT protease that cleaves the bond between the two basic amino acids. Expected to be able.

Thus, on the basis of the above assumptions, A novel expression plasmid vP8D expressing a fusion protein comprising a portion of an aminoglycoside 3'-phosphortransase fused to an E. coli β-galactosidase derivative / V8 protease derivative fusion protein was constructed. Note that the V8 protein derivative (hereinafter referred to as V8D protein) encoded by the plasmid lacks 8 amino acids at the C-terminus as compared to the V8 protease derivative II described above, and the N-terminus and C- The terminus is fused to a portion of the E. coli β-galactosidase derivative and aminoglycoside 3′-phosphotransferase via the R6 linker peptide.

E. coli JM101 containing pV8D was cultured and induced by IPTG, and from SDS PAGE, it was found that about 60 kd of the fusion protein thus expressed formed an insoluble inclusion body in the cell.

Culture cells are then crushed and the crushed material is centrifuged to separate inclusion bodies containing the fusion protein and then dissolved with denaturing agents such as urea, guanidine, hydrochloride or surfactant.

In an embodiment of the invention, the inclusion bodies are dissolved in 8M urea and the mixture is diluted to 4M urea and then the whole is incubated at 37 ° C for 2 hours. Under these conditions, the E. coli-specific ompT protease cleaves the fusion protein and, upon SDS PAGE analysis, each of the β-galactosidase derivatives, the V8D protein and the aminoglycoside 3'-phosphotransferase protein, respectively. It was confirmed to provide the corresponding 12kDa, 26kDa and 22kDa bands.

On the other hand, when the same experiment was carried out using the ompT-defective mutant E. coli W3110M25 as a host, the above-mentioned band was not detected, which was the protease that specifically cleaved the fusion protein, which was the unique ompT protease. Shows. In addition, to confirm that the fusion protein was specifically cleaved with the ompT protease, a 26kDa band corresponding to the V8D protein was extracted from the SDS-PAGE gel and its N-terminal amino acid sequence was measured. As a result, it was confirmed that the arginine-arginine bond in the R6 linker-peptide was cleaved.

Thus, both the fusion protein and the ompT protease are present in the inclusion body precipitated by centrifugation, and after solubilization of the inclusion body with the 8M element, the ompT protease is completely active in the presence of the 4M element and correctly cleaves the expected site of the amino acid sequence. It was first discovered by the inventors.

Since the V8 protease derivative protein formed in the presence of urea has been denatured, the enzymatic activity of the product is believed to be very low. Thus, we performed a re-overlap of the V8D protein, if necessary, by reducing the denaturant concentration to determine whether an enzymatically active V8D protein could be obtained. After cleavage reaction with ompT protease, the sample is diluted 20-fold with 0.4 M potassium phosphate buffer (pH 7.5) and left overnight on ice. By this manipulation, about 20% of the V8D protein was re-overlaid and the enzyme activity was restored. After this manipulation, the samples are analyzed by SDS-PAGE. As a result, the main protein after re-overlap was the V8D protein.

The reason for this is that although there is a β-galactosidase derivative, part of the aminoglycoside 3'-phosphotransferase protein and E. coli-derived protein prior to redundancy, it has protease activity after redundancy. It is considered that the V8D protease hydrolyzed other accompanying proteins. This result is very advantageous for the purification of V8D protein after re-overlap.

The V8D protease activated as described above is then tested for whether it has the same substrate specificity as that of the native Staphylococcus aureus V8 protease. Substrates (e.g., fusion proteins comprising human calcitonin derivatives) are reacted with re-overlapping V8D proteases and natural enzymes at 30 ° C for 1 hour, cleaved with enzymes to generate peptide fragments from fusion proteins with high performance liquid chromatography. Analyze with As a result, the elution patterns of the peptide fragments produced by both enzymes were identical, indicating that the V8D protein prepared as described above had the same substrate specificity as that of the native enzyme.

s. In order to express the Aureus V8 protease intracellularly as an insoluble inclusion body, the protease should not act on the fusion protein. On the other hand, after re-overlap, the protease should exhibit its enzymatic activity. These apparently contradictory properties are required for V8 protease derivative proteins. For the V8D protease, a fusion protein comprising a native V8 protease moiety starting at the N-terminus and ending at the 212th amino acid is constructed, the C-terminus of the V8 protease moiety is elongated and amino glycoside 3'-phosphotransferase The fusion protein is then fused to a portion of the protein to form a fusion protein and then tested for the fusion protein as to whether it forms an inclusion body and whether it is reactivated by re-overlap.

That is, the present inventors have expressed expression plasmids pV8H, pV8F, pV8A, pV8D2 and pV8Q whose C-terminus express the fusion protein of the V8 protease extended by 2, 4, 6 and 8 amino acid residues, respectively, by PCR and gene cloning. It was constructed. These plasmids are used to transform E. coli JM101 to construct transformants. The resulting transformants are cultured and induced with IPTG. As a result, the transformant with the plasmid pV8D, pV8H or pV8F formed an inclusion body of the fusion protein, cleaved the fusion protein with the ompT protease, and then the re-overlapping enzyme was reactivated. On the other hand, the other transformants did not form inclusion bodies and the soluble fraction showed V8 protease activity after culturing the cultured cells. Thus, the E. coli V8 protease derivative protein must be fused with a protective polypeptide at its 215th amino acid, phenylalanine or an amino acid before 215th amino acid, to form an inclusion body of the fusion protein, followed by enzymatic cleavage and re-overlap of the fusion protein. It was found to follow.

Although the present invention has been described taking the V8 protease as an example of the desired polypeptide, the same principles and procedures are used for motiline, glucagon, corticosteroids (ACTH), corticotropin-releasing hormone (CRH), secretin, growth hormone, insulin , Growth hormone-releasing hormone (GRH), vasopressin, oxytocin, gastrin, glucagon-like peptide (GLP-1, GLP-2, 7-36 amide), cholecystokinin, angiogenic intestinal polypeptide (VIP), pituitary adenol Late cyclase activating polypeptide (pacap), gastrin releasing hormone, galanin, thyroid-stimulating hormone (TSH), progesterone-releasing hormone (LH-RH), calcitonin, parathyroid hormone (PTH, PTH (1-34) ), PTH (1-84), peptide histidine isoleucine (PHI), neuropeptide Y (nP.Y), peptide YY (P.YY), pancreatic polypeptide (PP), somatostatin, TGF-α, TGF- β, nerve growth factor, fibroblast growth factor, Raxine, prolactin, atrial sodium diuretic peptide (ANP), sodium diuretic peptide (BNP), sodium diuretic peptide (CNP), angiotensin, brain-derived nutrient factor (BDNF) and other enzymes (e.g. It can be applied to other target polypeptide or protein such as KEX2 endoprotease) to enzymatically produce the target polypeptide or protein as described above.

Thus, the present invention transforms a host cell with an expression vector containing a gene encoding a protective polypeptide and a fusion protein comprising the desired polypeptide, expresses the gene to produce a fusion protein, and then Provided is a method of producing a desired polypeptide, characterized in that the desired polypeptide is cleaved with a protease.

According to the invention, the fusion protein can be represented by the general formula (1) ALB or (2) ALBLC, where A and C are protective polypeptides, B is the desired polypeptide, and L is directed to a host cell specific protease. A linker peptide containing a substrate site recognized by the fusion protein, wherein the fusion protein is cleaved in the linker peptide L such that the desired polypeptide is obtained therefrom

According to a preferred embodiment of the invention, the polypeptide of interest is a biologically or physiologically active polypeptide, preferably an enzyme, more preferably a protease. In the most preferred embodiment of the present invention, the polypeptide of interest is a protease expressed as an inactive fusion protein in the host cell, the host cell is pulverized to separate the fusion protein and then solubilized with a denaturant, and then the linker peptide region is unique to the host cell. Cleavage with a protease yields the desired polypeptide from the fusion protein.

In another preferred embodiment, the polypeptide of interest is a proteolytic enzyme, the polypeptide of interest is expressed in the host cell as an insoluble fusion protein containing the polypeptide of interest bound to the protective polypeptide via a linker peptide, and the host cell is crushed to Isolates the fusion protein, solubilizes the fusion protein as a denaturant at a concentration where the host cell's native protease is not active, and then reduces the denaturant's concentration to the level at which the native protease's enzymatic activity is exhibited, thereby cleaving the linker peptide with the protease and fusion Mention is made of methods of producing the desired polypeptide, which obtain the desired polypeptide from the protein. In this case, during the isolation process after cell disruption, the native protease and the fusion protein are preferably coexist in the same fraction.

The protective polypeptide can be any polypeptide that can be expressed as part of a fusion protein containing the desired polypeptide, for example a polypeptide derived from E. coli β-galactosidase, amino of transposon 903 Polypeptides derived from glycoside 3'-phosphotransferase and the like can be used alone or together.

Linker peptides are peptides having a site specifically recognized by a host cell specific protease containing the expression vector for the polypeptide of interest. A preferred embodiment of the linker peptide is a polypeptide consisting of 2 to 50 amino acid residues and containing at least two basic amino acid residue pairs. The linker peptide may have a pair of basic amino acid residues at both its N- and C-terminus.

The denaturant can be any substance that solubilizes the fusion protein and is, for example, urea, guanidine hydrochloride, surfactants, and the like. Preferably urea is used and in this case the concentration of urea is preferably 1 to 8M. Urea concentration after solubilization of the fusion protein may be any concentration at which the host cell's native protease exhibits its enzymatic activity.

The native protease and the desired polypeptide are not limited in any way in the present invention related to the method of producing the polypeptide of interest in which the polypeptide of interest is expressed as a fusion protein and the fusion protein is cleaved into a host cell specific protease. That is, the native protease can be any protease that can process the fusion protein after the fusion protein is expressed as an insoluble protein, for example the E. coli ompT protease used in the Examples and the like. The polypeptide of interest may preferably be any polypeptide consisting of 20 to 800 amino acid residues, for example as shown in the Examples below. Aureus proteases and / or derivatives thereof.

According to the present method for production of the desired polypeptide using genetic recombination technology, in particular v8 protease derivative proteins can be produced in the E. coli expression system. That is, preferred embodiments for producing the desired polypeptide

(1) an E. coli host cell comprising a fusion protein comprising one or more protective polypeptides, a desired polypeptide and a linker peptide, wherein the protective polypeptide is a polypeptide derived from E. coli β-galactosidase And / or a polypeptide derived from an aminoglycoside 3'-phosphotransferase of transposon 903 origin, the polypeptide of interest is a derivative of the Staphylococcus aureus V8 protease, between the protective polypeptide and the desired polypeptide. The linker peptide has a substrate site that is specifically recognized by a host cell specific protease);

(2) expressing said gene in an E. coli host cell to produce a derivative of Staphylococcus aureus V8 protease as an inactive fusion protein;

(3) crushing the cells to separate fusion proteins and obtaining a fraction containing these cell-specific proteases, the E. coli ompT protease and the fusion protein;

(4) solubilizing the fusion protein with a denaturing agent; And

(5) obtaining the desired polypeptide from the fusion protein by cleaving the linker peptide with the protease by reducing the concentration of denaturant to the level at which the activity of the E. coli ompT protease appears.

After re-overlapping the V8 protease derivatives, the protein can be highly purified by conventional methods for protein purification, such as gel filtration, ion chromatography, hydrophobic chromatography. In addition, in the case of the V8 protease derivatives as shown in the examples, purification is very easy since the derivatives are the main proteinaceous components in the reaction mixture after the re-overlap reaction.

Example

The invention is then described in more detail in the following examples.

Example 1

s. Isolation of the Aureus V8 Protease Gene

The V8 protease gene is prepared by PCR method based on the reported nucleotide sequence. The three PCR primers shown in FIG. 1 (b) are synthesized by a DNA sequence analyzer (manufactured by Applied Biosystem). As shown in Figure 1, primers I, II and III correspond to regions of the V8 protease gene and primer I has an XhoI restriction site at its 5'-end and primers II and III have their 5'-end. Has a SalI restriction enzyme site. PCR is described in Jayaswal et al., J. Bacterial. 172: 5783-5788 (1990), using chromosomes prepared from Staphylococcus aureus V8 (ATCC 27733) and PCR primers described above. 2.5 units of Taq DNA polymerase were obtained from 1.0 μM primer, 1 μg chromosomal DNA, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl ₂ , 0.01% gelatin, 200 μM dNTP (dATP, dGTP, dCTP and dTTP). Mixture), and PCR is carried out for 30 cycles for 1 minute at 90 ° C, 2 minutes at 72 ° C, and 2 minutes at 55 ° C.

As a result, the genes for the mature V8 protease (protease gene derivative I, 0.8 kb) containing the repeat sequence but not the prepro sequence are obtained by primers I and II and contain neither the propro sequence nor the repeat sequence. Gene for V8 protease (V8 protease gene derivative II, 0.7 kb) was obtained by primers I and III. These genes are then electrophoresed, purified with SUPREP-2 (manufactured by Takara Shuzo), and then cleaved with restriction enzymes XhoI and SalI to yield V8 protease derivative gene fragments I and II with sticky ends XhoI and SalI.

Example 2

Construction of the expression vectors pV8RPT (+) and pV8RPT (-) and expression of V8 protease derivatives

Plasmid pG97S4DhCT [G] R6 used in this Example is a plasmid that efficiently expresses a fusion protein containing an E. coli β-galactosidase derivative and a human calcitonin precursor (hCT [G]), and plasmid pBR322 and plasmid It can be constructed from pG97S4DhCT [G] (see Japanese Unexamined Patent Publication No. 5-328992, EP 528686 and Fig. 2). E. coli W3110 containing plasmid pG97S4DhCT [G] was named E. coli SBM323 and deposited under the Budapest Treaty (National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, 1-3 Higashi, 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), which was deposited on August 8, 1991 as International Deposit No. FERM BP-3503.

To express the V8 protease genes I and II obtained by PCR, plasmid pG97S4DhCT [G] R6 was digested with XhoI and SalI, and DNA fragments (3.1 kb) lacking the human calcitonin precursor gene were subjected to agarose gel electrophoresis. Manufactured by This DNA fragment was linked to a V8 protease gene fragment having XhoI and SalI sticky ends as prepared above using T4 DNA ligase and the plasmid pV8RPT (+) containing the V8 protease gene derivative I using the linkage product. And E. coli JM101 was transformed to construct plasmid pV8RPT (−) containing V8 protease gene derivative II (see FIG. 3). As a host for the plasmid, E. coli JM101 (eg, available from Takara Shuzo, Invitrogen Catalog No. c660-00, etc.) is used. The amino acid sequence of the fusion protein containing the V8 protease derivative and the β-galactosidase derivative expressed by the plasmid described above is shown in FIG. E. coli JM101 / pV8RPT (+) and E. coli JM101 / pV8RPT (-) were added in 100 ml LB medium (0.5% yeast extract, 1.0% tryptone, 0.5% NaCl) until absorbance OD 660 reached 1.0. Incubate separately at ° C. and induce gene expression by adding isopropyl thiogalactropyranoside (IPTG) to a final concentration of 2 mM. After addition, the cells are further incubated for an additional 2 hours, the culture is centrifuged to recover the microbial cells, and then suspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at a concentration of OD 660 = 5.

The cell suspension is treated with a sonicator (cell shredder, Tosho Denski K.K), the milled material is centrifuged at 12,000 rpm for 5 minutes to remove the insoluble fraction, and the supernatant thus obtained is used as crude enzyme preparation. Activity of the V8 protease was measured using a synthetic substrate (Z-Phe-Leu-Glu-4-nitranylide; Boehringer Mannheim) and 940 μg 100 mM Tris-HCl (pH 8.0) buffer and 20 μg 10 mM in DMSO Z-Phe-Leu-Glu-4-nitranylide solution is mixed and 40 μl of crude enzyme solution is added to this mixture. The mixture is incubated at room temperature for 5 minutes and the absorbance of the reaction mixture is measured at 405 nm using Hitachi spectrophotometer U-3200.

As a result, the crude enzyme solution prepared from E. coli JM101 / pV8RPT (+) and E. coli JM101 / pV8RPT (−) cells provided enzymatic activity of 8 μg / ml, which contained β-galactosidase. And the enzyme in the form of a fusion protein with a missing prepro sequence shows enzymatic activity. In addition, products lacking the C-terminal repeat sequence, encoded by pV8RPT (-), also exhibit enzymatic activity, indicating that the repeat sequence is not essential for enzymatic activity.

Low production rate of V8 protease derivatives I and II by E. coli JM101 / pV8RPT (−) and E. coli JM101 / pV8RPT (+) and its band detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) Of course, its purification is also difficult. Given the fact that cell growth is arrested by the addition of IPTG and that the content of intracellular polymer proteins induced is lower than in non-induced cells, the low production rate is due to the lethal toxicity of the intracellularly expressed V8 protease. It is feed.

Example 3

Construction of Expression Vector pV8D

As can be seen from the above, since the V8 protease fused with the β-galactosidase derivative at the N-terminus still has its own enzymatic activity, the V8 protease is ineffective by fusion only at its N-terminus. It cannot be activated. Accordingly, we attempted to inactivate the V8 protease by further fusing the C-terminus of the enzyme with the portion of the aminoglycoside 3'-phosphotransferase. An EcoRV site located before the repeat sequence was used to fuse the C-terminus of the V8 protease.

Plasmid pV8D encoding the V8 protease derivative is constructed according to the procedure shown in FIG. That is, BglII-SalI fragments (3.0 kb) and EcoRV-BglII fragments (0.7 kb) were prepared from pV8RPT (−), and NarI-SalI fragments (0.2 kb) were prepared from pG97S4DhCT [G] R10 and these three DNAs Ligation of fragments yields pV8hCT [G]. Note that pG97S4Dh [G] R10 can be constructed from plasmid pBR322 and plasmid pG97S4DhCT [G] following the same method for plasmid pG97S4DhCT [G] R6 described above (Japanese Unexamined Patent Publication No. 5-328992, EP528686, FIG. 2).

Subsequently, the hCT [G] region (0.1 kb BstEII-Sal fragment) in pV8hCT [G] obtained as described above was replaced with pUC4K [Vieira, J. and Messing, J., Gene 19, 259 (1982), Pharmacia Ca. No. 27-4958-01] constructs pV8D by replacing with 0.8 kb SmaI-SalI fragments containing the aminoglycoside 3'-phosphotransferase gene region. The amino acid sequence of the fusion protein (V8D fusion protein) containing the V8 protease derivative expressed by plasmid pV8D is shown in FIG.

The fusion protein contains a V8 protease linked to a β-galactosidase derivative and a portion of aminoglycoside 3′-phosphotransferase at its N- and C-terminus, respectively, via a R6 linker. R6 linker is a peptide bond between R-R. Has the sequence RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1) that can be cleaved with the ompT protease of E. coli.

Example 4

According to the conventional method, E. coli transformed with pV8D. E. coli JM101 / pV8D is incubated at 37 ° C. in 100 ml LB medium until absorbance 0D 660 reaches 0.6 and IPTG is added to a final concentration of 100 mM to induce the production of fusion protein V8D. After addition, the cells are further incubated for 2 hours, and the culture solution is recovered by centrifugation. this. E. coli JM101 / pV8RPT (+) and E. coli. Unlike in the case of E. coli JM101 / pV8RPT (-), cell growth was not terminated by induction of expression of the fusion protein in this strain, and V8 protease activity was not detected in the cells.

In addition, intracellular formation of inclusion bodies was observed under a microscope. The results of 16% SDS-PAGE on cells and 16% SDS-PAGE on insoluble and soluble fractions obtained by sonicating the cells before and after induction are shown in FIG. Because inclusion bodies were formed, large amounts of 60 kDa V8D fusion protein were contained in the induced cells and insoluble fractions. Since the V8 protease derivative fused with a portion of the aminoglycoside 3'-phosphotransferase at its C-terminus cannot take its natural conformation, it is inactive and therefore does not inhibit cell growth, thus the expression of the inclusion body It is assumed that the level formed is reached. Note that in addition to the 60 kDa V8D fusion protein, 27 kDa protein was detected in the insoluble fraction, and the 27 kDa protein was found to contain an amino acid sequence containing the 282th methionine and a lower region as a fragment of the V8D fusion protein.

Example 5

Treatment of V8D Fusion Proteins with ompT Protease

The microbial cells obtained by culturing as in Example 4 are suspended in 10 ml of TE buffer and triturated by sonication. After this treatment, the inclusion body is recovered by centrifugation. The resulting inclusion body is resuspended in 10 ml of deionized water and the suspension is centrifuged to wash the inclusion body. Inclusion bodies are diluted with deionized water until the 0D 660 value reaches 100 and 150 μl of reaction mixture is taken. To 150 μl of sample, 25 μl of 1M Tris-HCl (pH 8.0), 2.5 μl of 1M dithiothreitol (DTT) and 120 mg of urea were solubilized, and the resulting solution was added deionized water to a total volume of 500 Allow to μl. The resulting solution is heated at 37 ° C. for 2 hours.

The 16% SDS-PAGE results before and after heating are shown in FIG. 8A. As can be seen from FIG. 8A, the sample after heat treatment corresponds to the part of β-galactosidase derivative, V8 protease derivative and aminoglycoside 3'-phosphotransferase having molecular weight of 12 kDa, 26 kDa and 22 kDa, respectively. A band was provided. On the other hand, the protease-deficient strain E. coli. E. coli W3110M25 by Sugimura, K. (1987) Biochem. Biophys. Res. Commun. 153, 753-759], express the V8D fusion protein and the resultant obtained by treating the inclusion body according to the same method as described above is shown in Figure 8B. In this case, the three bands described above were not detected in SDS-PAGE, so it was determined that treatment of this fusion protein was specifically cleaved by the ompT protease. Sugimura, K. and Nishihara, T. (1988) J. Bacteriol., 170, 5625-5632.

In addition, the 26-kDa band was cut from the SDS-PAGE to measure the N-terminal amino acid sequence of the fragment, and the binding between the RRs of the R6 sequence (RLYRRHHRWGRSGSPLRAHE) (SEQ ID NO: 1) was cleaved. It was confirmed that it was specifically performed by the ompT protease. During the aforementioned manipulations, the solubilization of inclusion bodies is carried out in the presence of 8M elements and the treatment is carried out in the presence of 4M elements, so that the ompT protease is specifically resistant to such high concentrations of elements and solubilizes solubilized proteins from inclusion bodies. It showed for the first time that it could be cut.

Example 6

Re-overlap of Recombinant V8 Protease (V8D)

Samples after treatment are diluted 20-fold with 0.4 M potassium phosphate buffer (pH 7.5) and allowed to stand on ice overnight. By this manipulation, the recombinant V8 protease (V8D) was re-overlapping and exhibited activity corresponding to 30 μg / ml as measured above. The re-overlap rate was about 20%. 16% SDS-PAGE results before and after re-overlap are shown in FIG.

Upon re-overlap, the recombinant V8 protease (V8D) is a β-galactosidase derivative, a protein derived from aminoglycoside 3′-phosphotransferase and other E. coli. It acts as a potent protease for E. coli-derived proteins, so these proteins are degraded and destroyed. Thus, the sample after re-incorporation contains the recombinant V8 protease (V8D) as the main protein, and thus the purification process after re-overlap can be extensively simplified.

The recombinant V8 protease thus obtained (V8D), compared to the native V8 protease, lacked 56 amino acid residues at the C-terminus due to the construction of the gene, but retained its own activity, which is necessary for enzyme activity. Not.

Example 7

Substrate Specificity of Presynthetic V8 Protease Obtained by Reoverlap from Inclusion Body

In order to compare the substrate specificity of the recombinant V8 protease obtained by re-overlap and the substrate specificity of the native V8 protease, the two proteases act as a substrate to release the fusion protein (hCT [G]) of the calcitonin precursor to release hCT [G]. The experiment was performed. The calcitonin fusion protein used in the experiment contained β-galactosidase derivatives (108 amino acid residues) and hCT [G] linked via a linker with glutamic acid, and the native V8 protease degraded the peptide bonds of the carboxy side of glutamic acid. To release hCT [G]. Note that pG97S4DhCT [G] R4 may be mentioned as a plasmid encoding the fusion protein (Japanese Unexamined Patent Publication No. 5-328992, EP528686).

The amount of recombinant V8 protease (V8D) corresponding to the activity of 1.2 μg native V8 protease was measured in 1 ml of solution containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 5 mM DTT, 2M urea and 10 mg / ml human calcitonin fusion protein. And the mixture is reacted at 30 ° C. for 1 hour and then analyzed by high performance liquid chromatography eluting with 0.1% trifluoroacetic acid (TFA) and 0.1% TFA / 50% acetonitrile. Note that the recombinant V8 protease (V8D) is used after re-overlap without further purification and commercially available natural V8 protease is used as a control.

The elution pattern of high performance liquid chromatography is shown in FIG.

The cleavage patterns of the recombinant V8 proteases and the native V8 proteases for the human calcitonin fusion protein were identical, confirming that the substrate specificities of both proteases are the same.

Example 8

Study of C-terminal fusion site of V8 protease

In order to construct a fusion protein of the V8 protease whose C-terminus is extended by 2, 3, 4, 6 and 8 amino acid residues, respectively, as compared to the V8D fusion protein, the PCR primers shown in FIG. 11 are synthesized. A plasmid pV8F encoding a fusion protein (V8F fusion protein) extended by three amino acid residues was constructed as follows.

That is, the V8 protease gene was amplified using 0.1 µg of pV8RPT (-) constructed in Example 1 using primer b and primer I shown in FIG. 1 (b), and the amplified DNA fragment was expressed by EcoRI and Cleavage with SacI produces a 0.1 kb gene fragment. Meanwhile, the R6 linker sequence and aminoglycoside 3'-phosphotransferase gene region were amplified using primer g, primer h and 0.1 μg of pV8D as template DNA, and the amplified DNA fragment was digested with EcoT22I and SacI to 0.3 Prepare the kb gene fragment. Note that the PCR is performed under the same conditions as described in Example 1. PV8F was constructed by linking the 0.1 kb and 0.3 kb gene fragments obtained as described above and the EcoRI-EcoT22I fragment (4.2 kb) of pV8D (see also FIGS. 12 and 14).

Plasmids pV8H, pV8A, pV8D2 and pV8Q were constructed using primers g and primers a, c, d and e, respectively, following the same procedure as above (except for NdeI instead of SacI for pV8H construction). . The combination of the primer and template DNA used in the construction of the plasmid described above is as follows.

pV8H: PCR product obtained by combining primer a, primer I and pV8RPT (-) and primer f, primer h and pV8D;

pV8A: PCR product obtained by combining primer c, primer I and pV8RPT (-) and primer g, primer h and pV8D;

pV8D2: PCR product obtained by the combination of primer d, primer I and pV8RPT (-) and primer g, primer h and pV8D;

And

pV8Q: PCR product obtained by combining primer e, primer I and pV8RPT (-) and primer g, primer h and pV8D.

These plasmids produce a fusion protein containing a V8 protease region elongated by 2, 4, 6 and 8 amino acid residues, respectively, as compared to the V9D fusion protein shown in Example 4 (see also 13 and 14). ).

Using these plasmids, E. coli. E. coli JM101 is transformed and tested for expression of each fusion protein according to the same method as described in Example 4. The results are shown in FIG. Inclusion bodies were formed from pV8H, pV8F and pV8D, and the inclusion bodies thus obtained are converted to re-overlapped active V8 protease according to the same method as described in Example 5. PV8A, pV8D2 and pV8Q did not form inclusion bodies and V8 protease activity was detected in the soluble fraction. For these plasmids, it is believed that no inclusion bodies are formed because the expressed fusion protein has protease activity and inhibits the growth of host cells. In other words, in order to express the V8 protease as an inactive fusion protein, it is important to fuse the V8 protease with a protective polypeptide at its 215th phenylalanine or at an amino acid (near the N-terminus) preceding this phenylalanine and the V8 protease is 215 When fused at the amino acid (near the C-terminus) after the first phenylalanine, growth was inhibited and expression was reduced because the V8 protease residues produced a fusion protein that forms a natural conformation showing protease activity.

According to the present invention, large amounts of the desired polypeptide can be produced at low cost. In particular, according to the present invention, a large amount of Staphylococcus aureus V8 protease is dependent on the method of genetic recombination. It can be produced efficiently and at low cost using a safe host such as E. coli.

1 (a) and 1 (b) are (a) construction of Staphylococcus aureus (S. aureus) V8 protease-encoding gene and (b) PCR used to clone the gene of the present invention. It is a figure which shows the nucleotide sequence of a primer.

2 is a construction process diagram of pG97S4DhCT [G] R6 and pG97S4DhCT [G] R10.

3 is a process chart of the construction of plasmids pV8RPT (+) and pV8RPT (−).

4 (a) and 4 (b) are amino acid sequence diagrams encoded in plasmids pV8RPT (+) and PV8RPT (−).

5 is a construction process diagram of plasmid pV8D.

6 is an amino acid sequence diagram of the fusion protein encoded in plasmid pV8D.

7 is an electrophoresis result showing that the fusion protein forms inclusion bodies and migrates to insoluble fractions.

8A and 8B are electrophoresis results showing that the fusion protein V8D is cleaved by host-derived protease ompT to release the V8 protease.

9 is an electrophoresis result showing the refolding of the V8 protease liberated by the ompT protease.

10A and 10B show (A) recombinant V8 protease and (B) S. obtained by the method. A comparison chart between products formed from fusion proteins containing human calcitonin precursors by cleaving the fusion proteins with the V8 protease obtained from S. aureus.

11 is a nucleotide sequence diagram of the primers used for the construction of DNA (pV8H, pV8F, pV8A, pV8D2 and pV8Q) encoding various fusion proteins.

12 is a construction flow diagram of plasmids pV8H, pV8F, pV8A, pV8D2 and pV8Q.

Figure 13 shows the C-terminal amino acid sequence diagram of the V8 protease encoded in plasmids pV8D, pV8H, pV8F, pV8A, pV8D2 and pV8Q and formation of inclusion bodies from the expression product (fusion protein) of the plasmid.

Figures 14 (a), 14 (b) and 14 (c) are amino acid sequences of the polypeptide of interest produced by the method of the present invention.

Claims (34)

(1) transforming a host cell with an expression vector containing a gene encoding a fusion protein comprising a polypeptide of interest and a protective polypeptide; (2) culturing the transgenic host cell to express the gene to produce a fusion protein that is insoluble and deposited intracellularly as inclusion bodies; (3) crushing host cells to separate inclusion bodies, and (4) A method for producing a target polypeptide, comprising the steps of cleaving the target polypeptide from the fusion protein with a native protease from the crushed host cell itself, isolated with inclusion bodies, to produce the desired polypeptide. The method of claim 1, wherein the fusion protein is of the formula (1) ALB or (2) ALBLC, wherein A and C are protective polypeptides, B is the polypeptide of interest, and L is by a host cell-specific protease. And a fusion protein is cleaved at the linker peptide L region such that the desired polypeptide B is obtained. The method of claim 1 or 2, wherein the polypeptide of interest is a physiologically active polypeptide. The physiologically active polypeptide of claim 3, wherein the physiologically active polypeptide is motiline, glucagon, corticosteroids (ACTH), corticotropin-releasing hormone (CRH), secretin, growth hormone, insulin, growth hormone-releasing hormone (GRH). , Vasopressin, oxytocin, gastrin, glucagon-like peptide (GLP-1, GLP-2, 7-36 amide), cholecystokinin, vascular functional intestinal polypeptide (VIP), pituitary adenoleate cyclase activating polypeptide (pacap ), Gastrin releasing hormone, galanin, thyroid-stimulating hormone (TSH), luteinizing hormone-releasing hormone (LH-RH), calcitonin, parathyroid hormone [PTH, PTH (1-34), PTH (1-84), Peptide histidine isoleucine (PHI), neuropeptide Y (nP.Y)], peptide YY (P.YY), pancreatic polypeptide (PP), somatostatin, TGF-α, TGF-β, nerve growth factor, fibroblast Growth factor, relaxin, prolactin, sodium diuretic peptide, angiotensin and Method selected from the group consisting of brain-derived nutrient factors. The method of claim 4, wherein the natriuretic peptide is selected from the group consisting of ANP, BNP and CNP. The method of claim 3, wherein the physiologically active polypeptide is an enzyme. The method of claim 5, wherein the enzyme is KEX2 endopeptidase. The method of claim 6, wherein the enzyme is a proteolytic enzyme. The method of claim 6, (a) expressing a polypeptide of interest as an inactive fusion protein in a host cell; (b) crushing said host cell; (c) isolating the fusion protein; (d) solubilizing the fusion protein with a denaturing agent; And (e) cleaving the linker peptide region with a host cell-specific protease such that the desired polypeptide is obtained from the fusion protein. 10. The method of claim 9, wherein step (e) is performed by reducing the concentration of denaturant. The method of claim 9, wherein the host cell-specific protease and the fusion protein are present in the same fraction during the separation process after cell disruption. The method of claim 9, wherein the denaturing agent for solubilization of the fusion protein is selected from the group consisting of urea, guanidine hydrochloride and surfactant. The method of claim 12, wherein the denaturing agent is 1-6M urea. The host cell-specific protease of claim 1, wherein the host cell-specific protease is E. coli. E. coli ompT protease. The method of claim 9, wherein the host cell-specific protease is E. coli. E. coli ompT protease, wherein the fusion protein is cleaved with the protease in a solution containing 1-6M urea. The method of claim 2, wherein the linker peptide has a site specifically recognized by a host cell-specific protease containing the expression vector for the polypeptide of interest. The method of claim 2, wherein the linker peptide consists of 2 to 50 amino acid residues and contains one or two pairs of basic amino acids. The method of claim 2, wherein the linker peptide has basic amino acid pairs at its N- and C-terminus. The method of claim 16, wherein the linker peptide has the amino acid sequence RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1). 20. The method of any one of claims 1, 2 and 16-19, wherein the polypeptide of interest has 20 to 800 amino acid residues. The method of claim 8, wherein the protease is Staphylococcus aureus V8 protease or derivatives thereof. The method of claim 21, wherein the Staphylococcus aureus V8 protease derivative has an amino acid sequence shown in FIG. 4 or 6 as dotted lines or an amino acid sequence shown in FIG. 14. 20. The method of any one of claims 1, 2 and 16-19, wherein the protective polypeptide is E. coli. A polypeptide derived from E. coli β-galactosidase and / or a polypeptide derived from aminoglycoside 3′-phosphotransferase of transposon 903 origin. 20. The method of any one of claims 2 and 16-19, wherein the protective polypeptide A is E. coli. A polypeptide derived from E. coli β-galactosidase, and protective polypeptide C is a polypeptide derived from aminoglycoside 3′-phosphortransferase of transposon 903 origin. The method of claim 12, wherein the desired polypeptide cleaved by the ompT protease in the presence of the denaturing agent is refolded by decreasing the concentration of the denaturing agent to obtain the desired polypeptide of activity. The method of claim 25, wherein the polypeptide of interest is a derivative of Staphylococcus aureus V8 protease. (1) an Escherichia coli host cell comprising a fusion protein comprising one or more protective polypeptides, a target polypeptide and a linker peptide, wherein the protective polypeptide is an E. coli β-galactosidase. Polypeptides derived from and / or polypeptides derived from aminoglycoside 3′-phosphotransferases of transposon 903 origin, the polypeptide of interest is a derivative of Staphylococcus aureus V8 protease, and a protective polypeptide And a linker peptide between the target polypeptide and the desired polypeptide has a substrate site that is specifically recognized by the host cell-specific protease). (2) the gene. Expression in E. coli host cells to produce derivatives of Staphylococcus aureus V8 protease as inactive fusion proteins; (3) Crushing the cells to separate the fusion protein, E. coli, such a cell-specific protease Obtaining a fraction containing E. coli ompT protease and a fusion protein; (4) solubilizing the fusion protein with a denaturing agent; And (5) Lee. A method for producing a polypeptide of interest, comprising the step of obtaining a target polypeptide from a fusion protein by cleaving the linker peptide with the protease by reducing the concentration of denaturant to the level at which the activity of E. coli ompT protease appears. The method of claim 27, wherein the denaturing agent for fusion protein solubilization is selected from the group consisting of urea, guanidine hydrochloride, and surfactant. 29. The method of claim 28, wherein the concentration of urea is between 1 and 8 M. The method of claim 29, wherein the fusion protein is e. The concentration of urea during cleavage with E. coli ompT protease is about 4M. 31. The method of any one of claims 27-30, wherein the linker peptide consists of 2 to 50 amino acid residues and contains a pair of two basic amino acids either at the linker peptide N- or C-terminus or in the linker peptide. 31. The method of any one of claims 27-30, wherein the linker peptide has the amino acid sequence RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1). 31. The method according to any one of claims 27 to 30, wherein the polypeptide of interest has an amino acid sequence underlined in FIG. 4 or 6 or an amino acid sequence as shown in FIG. 14 of the Staphylococcus aureus V8 protease. A derivative. 31. The method of any one of claims 27-30, further comprising (6) re-overlapping the desired polypeptide obtained in step (5) to obtain the active polypeptide of interest.