JP3995279B2 - Protein production method - Google Patents

Description

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【図面の簡単な説明】
【図１】図１は、スタフィロコッカス・アウレウス（Staphylococcus aureus）V8プロテアーゼ遺伝子の構成（ａ）、及び本発明の遺伝子のクローニングに用いたＰＣＲ用プライマーの塩基配列（ｂ）を示す図である。
【図２】図２は、pG97S4DhCT[G]R6 及びpG97S4DhCT[G]R10の作製過程を示す図である。
【図３】図３は、プラスミドpV8RPT（＋）及びpV8RPT（−）の作製過程を示す図である。
【図４】図４はプラスミドpV8RPT（＋）及びpV8RPT（−）中にコードされている融合蛋白質のアミノ酸配列を示す図である。
【図５】図５は、プラスミドpV8Dの作製過程を示す図である。
【図６】図６は、プラスミドpV8D中にコードされている融合蛋白質のアミノ酸配列を示す図である。
【図７】図７は、本発明の融合蛋白質が封入体を形成して不溶性画分に移行することを示す電気泳動図であり、図面に代る写真である。
【図８】図８は、V8D 融合蛋白質が、宿主由来のプロテアーゼompTにより開裂されてＶ８プロテアーゼを遊離させることを示す電気泳動図であって、図面に代る写真である。
【図９】図９は、ompTプロテアーゼにより遊離したＶ８プロテアーゼのリフォルディングを示す電気泳動図であり、図面に代る写真である。
【図１０】図１０は、ヒトカルシトニン前駆体を含む融合蛋白質を、本発明の方法により得た組換えＶ８プロテアーゼ（Ａ）又はＳ．アウレウスからのＶ８プロテアーゼ（Ｂ）により切断した場合の生成物を比較したチャート図である。
【図１１】図１１は、種々の融合蛋白質をコードするDNA を含むプラスミド（pV8H，pV8F，pV8A，pV8D2 及びpV8Q）の作製において使用したプライマーの塩基配列を示す図である。
【図１２】図１２は、プラスミドpV8H，pV8F，pV8A，pV8D2 及びpV8Qの作製の過程を示す図である。
【図１３】図１３は、プラスミドpV8D，pV8H, pV8F，pV8A，pV8D2 及びpV8Q中にコードされているＶ８プロテアーゼのＣ−末端アミノ酸配列、及び各プラスミドの発現生成物（融合蛋白質）による封入体の形成の有無を表示した図である。
【図１４】図１４は本発明の方法により製造される目的ポリペプチドのアミノ酸配列の例を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a target polypeptide using a gene recombination technique. Preferably, the target polypeptide is a bioactive polypeptide, for example an enzyme such as a protease. As an example, the target polypeptide is Staphylococcus aureus (Staphylococcus  aureus) It relates to a method for producing the polypeptide which is a V8 protease derivative.
[0002]
As a more specific example, Staphylococcus aureus (Staphylococcus  aureus) V8 protease derivative derived from V8 strain is expressed as an insoluble fusion protein, and the V8 protease derivative is cleaved from the fusion protein with endogenous ompT protease derived from E. coli in the presence of a denaturing agent, and refolded if necessary. To produce an active V8 protease derivative.
[0003]
[Prior art]
Staphylococcus aureus (Staphylococcus  aureus) (S. Aureus (S.  aureus)) V8 protease isS.  aureus  V8 strain is a kind of serine protease secreted into the medium. This enzyme was developed in 1972 by Drapeau, G. R. et al.S.  aureus  Isolated and purified as a kind of serine protease that specifically cleaves the C-terminal side of glutamic acid and aspartic acid secreted into the medium of strain V8 (Jean Houmard and Gabriel R. Drapeu (1972) Proc. Natl. Acad. Sci USA 69, 3506-3509), Cynthia Carmona et al. Revealed the DNA base sequence of this enzyme in 1987 (Cynthia Carmona and Gregory L. Gray (1987) Nucleic Acids Res. 15, 6757).
[0004]
This enzyme is considered to be secreted as a mature protein after being expressed as a precursor consisting of 336 amino acid residues and then removing 68 prepro sequences from the N-terminus. Furthermore, it is known that this enzyme has a repeat (repeat) sequence of proline-aspartate-asparagine in the C-terminal region (amino acid numbers 221 to 256). It is unclear whether this repetitive sequence is necessary for enzyme activity, and Gray et al. Think that it functions when present as an inactive enzyme before it is secreted. Yes.
[0005]
In spite of insufficient functional analysis as an enzyme, the enzyme specifically cleaves the peptide bond on the C-terminal side of glutamic acid and aspartic acid, and is therefore widely used for determining the amino acid sequence of proteins. In addition, since this enzyme acts on the substrate even in the presence of urea (about 2M), solubilize the fusion protein expressed in large quantities in the bacterial body as an insoluble fusion protein by genetic recombination technology, and then act on this enzyme. It is also used in the process of releasing the target peptide from the fusion protein.
[0006]
The present inventors have succeeded in efficiently producing human calcitonin using the above-described method for producing human calcitonin by gene recombination (Japanese Patent Laid-Open No. 5-328992). In an example in which human glucagon is expressed as a fusion protein in an E. coli expression system, S. aureus V8 protease is used to excise human glucagon from the fusion protein (Kazumasa Yosikawa et al. (1992) Journal of Protein Chemistry, 11, 517-525).
[0007]
As described above, this enzyme is purified from the culture solution of S. aureus V8 strain, despite the fact that it has been used for peptide production by gene recombination. (1) (2) It must be purified from bacteria considered to be pathogenic bacteria, and (3) it is expensive.
[0008]
[Problems to be solved by the invention]
Accordingly, the present invention is directed to the mass production of a polypeptide of interest, such as S. aureus V8 protease. Production of high-purity target polypeptides, such as S. aureus V8 protease, is very useful in research and industry, and large-scale industrial production of such polypeptides is highly desired.
[0009]
[Means for Solving the Problems]
In order to achieve the object of the present invention, a gene encoding a polypeptide of interest, for example, a S. aureus V8 protease gene is isolated from a S. aureus V8 strain, and a safe host cell, for example, By using E. coli, it is necessary to produce and purify the polypeptide, for example, an enzyme in a large amount and at a low cost.
[0010]
In general, in order to produce a target polypeptide or protein by a genetic engineering method, the polypeptide produced by the host should not adversely affect the survival and growth of the host, and further the expression of the target polypeptide or protein. In order to facilitate the isolation and purification of the expressed target polypeptide or protein, the target polypeptide or protein is produced as a fusion protein and accumulated in the host cell as an insoluble inclusion body. It may be preferable.
[0011]
However, a fusion protein containing the target polypeptide or protein does not always form an inclusion body. For this reason, in one embodiment of the present invention, the target polypeptide or protein is expressed as a fusion protein with other proteins and accumulated in the host cell as inclusion bodies. Protease inherently possessed by the host cell cannot act on the inclusion body, but after the inclusion body is isolated and lysed, the host-derived protease associated with the inclusion body acts on the fusion protein. Can be cleaved to release the desired polypeptide or protein. Thus, according to the present invention, the target polypeptide or protein can be efficiently produced.
[0012]
However, when the target polypeptide or protein is made into a fusion protein according to a conventional method, the fusion protein does not always form an inclusion body. In such a case, in the present invention, an inclusion body is formed by forming a fusion protein by linking a protective peptide to the N-terminal side and the C-terminal side of the target polypeptide or protein.
Therefore, in the present invention, a method for efficiently producing a large amount of S. aureus V8 protease in an Escherichia coli expression system using genetic engineering techniques is described as a specific example.
[0013]
First, the present inventors considered that an expression method by a fusion protein method having no enzyme activity is appropriate for producing a large amount of S. aureus V8 protease using an E. coli expression system. Because this enzyme is a proteolytic enzyme, if this enzyme is expressed directly in the cells, it is thought that the E. coli protein is degraded and the growth of the bacteria is stopped, and a large amount of S. aureus V8 protease cannot be obtained. It is.
Therefore, it is expressed as a fusion protein having no enzyme activity, and then the S. aureus V8 protease portion is excised from the fusion protein using another protease, and refolded if necessary to express the activity. We considered purifying V8 protease.
[0014]
Furthermore, as another protease used for excision from this fusion protein, the present inventors considered use of ompT protease which is considered to exist in the outer membrane of Escherichia coli. This is because it is considered to be very disadvantageous in terms of cost to newly add another enzyme to produce S. aureus V8 protease at low cost.
Production by E. coli using ompT protease is as follows: (1) The manufacturing process is simple, (2) There is no need to add another protease to the reaction system, and the cost is reduced. (3) E. coli expression system Consideration of contamination with V8 protease production host-derived protein (S. aureus-derived protein for commercial V8 protease) that may be mixed with V8 protease when cleaved with the V8 protease produced using Escherichia coli as a host. Needless to say, this is very advantageous from the standpoint of not having to.
[0015]
E. coli ompT protease is a protease that exists in the outer membrane fraction of E. coli and selectively cleaves between basic amino acid pairs (Keijiro Sugimura and Tatsuro Nishihara (1988) J. Bacteriol. 170, 5625-5632). Sugimura et al. Purified ompT protease, cleaved various peptides with ompT protease using 50 mM phosphate buffer (pH 6.0) at 25 ° C. for 30 minutes, arginine-arginine, lysine-lysine, arginine-lysine and It is reported that it is an enzyme that cleaves between basic amino acid pairs of lysine-arginine.
[0016]
However, it does not mention whether the ompT protease has activity in the presence of urea (a urea concentration of 2 M or more). Since ompT protease exists in the outer membrane, S. aureus V8 protease is produced as inclusion bodies in E. coli, and then the insoluble fraction containing inclusion bodies is separated by centrifugation after disruption of the cells. Proteases may also coprecipitate.
[0017]
Urea is added to the precipitate fraction thus obtained to solubilize the fusion protein of S. aureus V8 protease. If the activity of ompT protease can be maintained under these conditions, it may be possible to use ompT protease for excision of S. aureus V8 protease from the fusion protein, or the further excised S. aureus V8 protease will be activated. It was thought that S. aureus V8 protease could be produced in a large amount by a simple process if refolding was performed for conversion.
[0018]
The present invention will be described below.
From the gene sequence of the S. aureus V8 protease gene, a signal sequence (pre-sequence) necessary for secretion and a pro sequence with unknown function are present on the N-terminal side of the mature protein, and the above-described repeat sequence is present at the C-terminus. It has been reported. Therefore, preparation of gene I from N-terminal to C-terminal of mature protein and preparation of gene II lacking a repeat sequence with unknown function on the C-terminal side of mature protein were performed.
I don't know if this unknown sequence is necessary for enzyme activity, but if not, lowering the molecular weight of the fusion protein can increase the number of expressed proteins per cell and increase the amount of expressed protein. I thought it would be possible.
[0019]
Therefore, chromosomal DNA was isolated from Staphylococcus aureus strain V8 (ATCC27733) and PCR was performed to prepare two types of V8 protease derivative genes I and II. In order to express these derivatives, expression plasmids pV8RPT (+) and pV8RPT (-) were prepared using E. coli β-galactosidase derivatives as protective peptides for the fusion protein. In these expression plasmids, a linker gene containing an arginine-arginine residue that is a recognition-cleaving amino acid sequence of ompT protease as a linker peptide gene between the E. coli β-galactosidase derivative protein gene and the V8 protease derivative gene (I or II) (for example, The fusion protein into which the R6 linker gene) is inserted is expressed under the control of the lactose promoter.
[0020]
In the fusion protein designed in this way, after expressing the V8 protease derivative as an insoluble fusion protein, the fusion protein is solubilized using urea, and if the urea concentration is lowered, the ompT protease cleaves the fusion protein, and E. coli This is because it was thought that β-galactosidase derivative protein and V8 protease derivative protein could be separated.
An expression plasmid designed as described above was prepared, and expression was induced using IPTG (isopropyl-β-D-thiaglactopyranoside; hereinafter referred to as IPTG) using E. coli JM101 as a host. Both were not insoluble, and the enzyme activity was observed in the supernatant fraction after disrupting the cells.
[0021]
On the other hand, after the expression induction, the proliferation of the cells was remarkably reduced. Analysis by SDS polyacrylamide gel electrophoresis revealed that the intracellular protein was degraded by the enzymatic activity of the expressed V8 protease derivative. Therefore, this fusion protein expression method of E. coli β-galactosidase derivative and V8 protease derivative is (1) that the expressed protein has enzyme activity and causes growth inhibition of the host, and (2) the expression level of the fusion protein is very high. Since it was low, it became clear that it was not suitable as a production method of a V8 protease derivative.
[0022]
However, from these results, (1) the N-terminal pro sequence may not be involved in the folding of the V8 protease, and (2) the V8 protease derivative of the type having no C-terminal repeat sequence. Since it is also active in II, it was considered that this repeat sequence may not be necessary for V8 protease enzyme activity.
Next, the present inventors expressed V8 protease as an insoluble fusion protein in the E. coli expression system, and then solubilized with urea to release the V8 protease portion from the fusion protein in the presence of urea using proteolytic enzyme. In producing the V8 protease having the enzyme activity by folding, the following working hypothesis was set and the experiment was started.
[0023]
(1) When the aforementioned E. coli β-galactosidase derivative is fused to the N-terminal side of V8 protease, it does not become an insoluble fusion protein. Therefore, in such a case, it is considered that a protective peptide is further added to form an insoluble fusion protein, and the kanamycin resistance gene which is transposon 903 (Nucleic Acids Res. (1988) 16, 358-358) is considered as the protective peptide. ) Derived aminoglycoside 3′-phosphotransferase protein. That is, by adding a part of the aminoglycoside 3′-phosphotransferase protein to the C-terminus of the fusion protein consisting of E. coli β-galactosidase derivative and V8 protease via a linker peptide, insolubilization of the fusion protein is promoted, and inclusion bodies are formed. I thought about doing it.
[0024]
(2) In order to enzymatically release the V8 protease part from the produced fusion protein, the above-mentioned R6 linker is arranged on the N- and C-terminal sides of the V8 protease, and ompT is used to cleave between basic amino acid pairs. It was thought that V8 protease could be dissociated from the fusion protein by cleavage.
[0025]
Accordingly, construction of a new expression plasmid was started based on the above working hypothesis, and a plasmid expressing a fusion protein in which a part of aminoglycoside 3′-phosphotransferase was fused to E. coli β-galactosidase derivative-V8 protease derivative. pV8D was prepared. The V8 protease derivative (hereinafter referred to as V8D protein) encoded by this plasmid is one in which 8 amino acids have been deleted from the C-terminus of the aforementioned V8 protease derivative II. The N-terminal side and C-terminal side of this V8D Is fused to part of the E. coli β-galactosidase derivative and aminoglycoside 3′-phosphotransferase via an R6 linker peptide.
[0026]
When E. coli JM101 carrying pV8D was cultured and induced for expression by IPTG, it was revealed by SDS PAGE analysis that the expressed 60 kilodalton fusion protein formed insoluble inclusion bodies in the cells. Therefore, it was revealed that insoluble inclusion body formation occurs by fusing a part of the aminoglycoside 3'-phosphotransferase protein to the C-terminal side of the V8D protein.
Next, after disrupting the cells, centrifugation is performed to separate the inclusion bodies, and the fusion protein is solubilized using a denaturing agent. As the denaturing agent, urea, guanidine hydrochloride, a surfactant or the like can be used.
[0027]
In the examples of the present application, inclusion bodies were solubilized with 8 M urea, diluted to 4 M urea concentration, and incubated at 37 ° C. for 2 hours. The endogenous ompT protease of E. coli cleaves the fusion protein under these conditions to produce 12 KDa, 26 KDa, and 22 KDa bands corresponding to β-galactosidase derivative, V8D protein, and part of aminoglycoside 3′-phosphotransferase protein, respectively. This was revealed by SDS PAGE analysis.
[0028]
On the other hand, as a result of conducting the same experiment using W3110M25, which is an ompT-deficient mutant, as a host fungus, the above band cannot be detected, and it is confirmed that the fusion protein is specifically cleaved by endogenous ompT. It was revealed. Furthermore, in order to confirm that it was specifically cleaved by ompT, a 26 KDa band corresponding to the V8D protein was cut out from the SDS PAGE gel and the N-terminal amino acid sequence was determined. As a result, the R6 linker peptide arginine-arginine was determined. It was confirmed that cleavage occurred between the sequences.
[0029]
Therefore, the fusion protein and E. coli endogenous ompT protease are present in the inclusion body fraction of the precipitate produced by centrifugation, and are solubilized by a denaturant, particularly after solubilization of the inclusion body by 8M urea, in the presence of 4M urea. However, the present invention revealed for the first time that it has sufficient enzyme activity and can accurately cleave the expected amino acid sequence.
Since the V8 protease derivative protein produced in the presence of urea is in a denatured state, the enzyme activity is considered to be very low. Therefore, if necessary for the expression of activity, the concentration of the denaturing agent was lowered to refold the V8D protein, and it was examined whether a V8D protein having enzyme activity could be obtained.
[0030]
The sample after the ompT cleavage reaction was diluted 20-fold with 0.4 M potassium phosphate buffer (pH 7.5) and then left overnight in ice. By this operation, the V8D protein was refolded by about 20%, and the enzyme activity was recovered. When the sample after this operation was analyzed by SDS-PAGE, V8D protein was mainly present after refolding.
This is because the sample before refolding contains β-galactosidase derivative, a part of aminoglycoside 3′-phosphotransferase protein, and protein derived from E. coli, but after refolding, other proteins in which V8D protein having protease activity is mixed. This is considered to be due to disassembling. This result is very advantageous when purifying the V8D protein after refolding.
[0031]
Next, the V8D protein that has been activated by the above-described procedure is converted into the natural type.S.  aureus  We examined whether there was the same substrate recognition as V8 protease. A V8D protein refolded on a substrate (for example, a fusion protein of human calcitonin precursor) and a natural enzyme are reacted at 30 ° C. for 1 hour, and a peptide fragment generated from the fusion protein cleaved by the enzyme is subjected to high performance liquid chromatography. Analyzed. As a result, the peptide fragments generated from both enzymes showed the same elution pattern, and it was revealed that the V8D protein produced by the above-described method has the same substrate specificity as the natural type.
[0032]
In order for S. aureus V8 protease to be expressed as an insoluble inclusion body in cells, it is necessary that the fusion protein has no enzymatic activity as a proteolytic enzyme. Must have. This seemingly contradictory property is required for the V8 protease derivative protein. In the V8D protein, a fusion protein was prepared using the amino acid from the N-terminal to the 212th amino acid of the natural V8 protease, but the V8 protease part was further extended to the C-terminal side and a fusion protein with a part of the aminoglycoside 3′-phosphotransferase protein. Whether or not reactivation by inclusion body formation and refolding occurs was investigated.
[0033]
The present inventors have used plasmids (pV8H, pV8F, pV8A, pV8D2, and pV8Q) that express fusion proteins in which the C-terminal side of the above-mentioned V8 protease derivative is extended by 2, 4, 6 and 8 amino acids by PCR and gene cloning. Produced. E. coli strain JM101 was transformed with these plasmids to produce recombinants.
When the obtained strain was cultured and expression was induced using IPTG, only the strains having the plasmids pV8D, pV8H, and pV8F described above formed inclusion bodies of the fusion protein. The enzyme could be reactivated during folding. Other strains did not form inclusion bodies, and V8 protease activity was observed in the soluble fraction after disruption of the cells.
[0034]
Therefore, the V8 protease derivative protein is expressed by the fusion protein expression method to form an inclusion body, then enzymatically cleaved and refolded with the 215th amino acid from the N-terminal of S. aureus V8 protease protein. It became clear that it was necessary to fuse before a certain phenylalanine.
As described above, the present invention has been specifically described by taking the V8 protease as an example of the target polypeptide or protein, but by applying the same principle and technique to other target polypeptides or proteins, the desired target polypeptide can be obtained. Alternatively, the protein can be produced efficiently.
[0035]
Therefore, the present invention transforms a host cell with an expression vector having a gene encoding a fusion protein comprising a protective polypeptide and a target polypeptide, expresses the gene, and uses the host cell's endogenous protease to express the fusion protein. The present invention relates to a method for producing a target polypeptide, which is obtained by cleaving the target polypeptide.
[0036]
Furthermore, the present invention provides a fusion protein represented by the formula (1) A-L-B or formula (2) A-L-B-L-C (where A and C are protected polypeptides, and B is a target polypeptide. , L represents a linker peptide having a substrate that is specifically recognized by the host cell endogenous protease), the fusion protein is cleaved at the region of the linker peptide L and the target poly The present invention relates to a production method characterized by obtaining peptide B. Here, in addition to the aforementioned S. aureus V8 protease, the target polypeptide is, for example, a physiologically active peptide such as motilin, glucagon, corticosteroid, corticotropin releasing hormone, secretin, growth hormone, insulin, growth hormone secretion Hormone, Pazopressin, Oxytocin, Gastrin, Glucagon-like peptide (GLP-1), Glucagon-like peptide (GLP-2), Glucagon-like peptide (7-36 amide), Cholecystokinin, Vasoactive Intestinal polypeptide ( vasoactive intestinal polypeptide; VIP),
[0037]
Pituitary adenolate cyclase activating polypeptide, gastrin releasing hormone, galanin, thyroid stimulating hormone, luteinizing hormone releasing hormone, calcitonin, parathyroid hormone (PTH, PTH (1-34) PTH ( 1-84)), peptide histidine isoleucine (PHI), neuropeptide Y (neuropeptide Y), peptide YY (peptide YY), pancreatic polypeptide, somatostatin, TGF-α , TGF-β, nerve growth factor, fibroblast growth factor, relaxin, prolactin, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type Diuretic peptide (C-type natriuretic peptide; C NP), angiotensin, brain-derived neurotrophic factor (BDNF) and, for example, enzymes such as KEX2 endoprotease.
[0038]
Further, as a preferred embodiment of the present invention, when the target polypeptide is a physiologically active polypeptide, preferably the physiologically active polypeptide is an enzyme, and more preferably, the enzyme is a proteolytic enzyme. . In a most preferred embodiment as a specific example, the target polypeptide is a proteolytic enzyme, the target polypeptide is expressed in a host cell as an inactive fusion protein, the cell is disrupted, and the fusion protein is After separation, the target protein is obtained from the fusion protein by solubilizing the fusion protein with a denaturant that solubilizes the fusion protein, and then cleaving the linker peptide region with a host cell endogenous protease. A method is mentioned.
[0039]
In another preferred embodiment, when the target polypeptide is a proteolytic enzyme, the target polypeptide is expressed in the host cell as an inactive fusion protein, and the cell is disrupted to separate the fusion protein. The fusion protein is solubilized with a denaturing agent that solubilizes the fusion protein, and then the concentration of the denaturing agent is lowered to such an extent that endogenous protease activity derived from the host cell can be expressed. Examples include a method for producing a target polypeptide, which is obtained from a fusion protein by cleaving with a protease. In any of the above cases, it is preferable that the endogenous protease derived from the host cell is present in the same fraction in the separation operation after disrupting the fusion protein and the cells.
[0040]
The protection polypeptide is not particularly specified as long as it can be expressed as a fusion protein with the protection polypeptide when the target polypeptide is expressed. For example, the polypeptide derived from E. coli β-galactosidase or the aminoglycoside derived from transposon 903 is used. Examples include 3′-phosphotransferase-derived polypeptides and the like, and if necessary, these polypeptides can be used in combination as a protective polypeptide.
[0041]
The linker peptide may be any linker peptide having a site that is specifically recognized by the endogenous protease of the host cell having the expression vector of the target polypeptide. As a preferable aspect of the linker peptide, for example, a basic amino acid pair sequence having one basic amino acid residue or continuous two basic amino acid residues is included in the linker peptide, and the linker peptide has 2 to 50 amino acids. A polypeptide consisting of residues is desirable, and the basic amino acid pair may be included in the N-terminal part and / or C-terminal part of the linker peptide.
[0042]
A denaturing agent for solubilizing the fusion protein need not be particularly specified, and examples thereof include urea, guanidine hydrochloride, and a surfactant. Preferably, urea is used, and the urea concentration in this case is desirably 1-8M. Moreover, the urea concentration after solubilizing the fusion protein is not particularly specified as long as the endogenous protease activity derived from the host cell can be expressed.
[0043]
The present invention also relates to a method for producing a target polypeptide, which is obtained by cleaving a target polypeptide from a fusion protein using an endogenous protease derived from a host cell. The peptide need not be particularly specified. That is, the protease only needs to be used for processing the target polypeptide or protein from the fusion protein after expressing the target polypeptide or protein as an insoluble fusion protein. Preferably, E. coli ompT protease and the like can be mentioned as shown in the following examples. The target polypeptide is preferably a polypeptide comprising 20 to 800 amino acid residues, and examples thereof include S. aureus V8 protease and / or its derivatives as shown in the following examples.
[0044]
As a result, it has been clarified that in the method for producing a target polypeptide using the gene recombination technique according to the present invention, a V8 protease derivative protein can be produced particularly in an E. coli expression system. That is, as a preferable embodiment in the method for producing the target polypeptide, the following production steps can be mentioned. That is;
[0045]
(1) The fusion protein comprises a protective polypeptide, a target polypeptide and a linker peptide, and the protective polypeptide is a β-galactosidase-derived polypeptide derived from E. coli and / or an aminoglycoside 3′-phosphotransferase-derived polypeptide derived from transposon 903. A linker peptide having a substrate in which the target polypeptide is a S. aureus V8 protease derivative and the linker peptide region between the protective polypeptide and the target polypeptide is specifically recognized by a host cell endogenous protease Transforming E. coli as a host cell with an expression vector having a gene encoding the fusion protein, which is a region,
[0046]
(2) expressing the fusion protein in E. coli with the S. aureus V8 protease derivative in an inactive state,
(3) After crushing the cells and separating the fusion protein, obtain a fraction containing E. coli ompT protease, which is an endogenous protease, and the fusion protein,
(4) solubilizing the fusion protein with a denaturant,
[0047]
(5) After solubilization, reducing the concentration of the denaturant to a state where the activity of the E. coli ompT protease is expressed, and cleaving the linker peptide region with the protease to obtain the target polypeptide from the fusion protein;
A step of producing refolding so that the target polypeptide becomes an active form, if necessary,
The production method characterized by the addition of
[0048]
As a method for purifying the protein after refolding the V8 protease derivative, the protein can be purified with high purity by, for example, usual protein purification operations such as gel filtration, ion chromatography, and hydrophobic chromatography. Furthermore, it goes without saying that the V8 protease derivative shown in the Examples is very easy to purify because the derivative is the main protein component of the reaction component at the end of the refolding reaction.
[0049]
【Example】
Hereinafter, the present invention will be described in detail in Examples.
Example 1.  S. aureus V8 Isolation of protease genes
In order to isolate the V8 protease gene by the PCR method, the gene was isolated by the PCR method based on the reported DNA nucleotide sequence. Three types of PCR primers having the sequence shown in FIG. 1 (b) were designed and synthesized using a DNA synthesizer (Applied Biosystems Model 392). Primers I, II and III correspond to the V8 protease gene region shown in FIG. 1 (a), and are provided with a Xho I or Sal I restriction enzyme site on the 5 ′ side.
[0050]
Staphylococcus aureus isolated and prepared by the method of Jayaswal, R. K. et al. (J. Bacteriol. 172: 5783-5788 (1990))Staphylococcus  aureus) PCR was performed using the V8 strain (ATCC27733) chromosome and these PCR primers. 50 μl reaction containing 1.0 μM primer, 1 μg chromosomal DNA, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.01% gelatin, 200 μM dNTP (mixture of dATP, dGTP, dCTP, dTTP) 2.5 units of Taq DNA polymerase was added to the solution, and PCR was performed 30 cycles of 94 ° C, 1 minute, 72 ° C, 2 minutes, 55 ° C, 2 minutes.
[0051]
As a result, a mature V8 protease gene (protease gene derivative I, 0.8 kb) that does not contain a prepro sequence and contains a repeat sequence is obtained by primer I and primer II, and the prepro sequence and repeat sequence are obtained by using primer I and primer III. A lacking V8 protease gene (V8 protease gene derivative II, 0.7 kb) was obtained. Next, these genes were subjected to agar gel electrophoresis, purified using SUPREP-2 (Takara Shuzo Co., Ltd.), cleaved with restriction enzymes XhoI and SalI, and V8 protease derivative genes with XhoI and SalI sticky ends. Fragments I and II were prepared.
[0052]
Example 2  Expression vector pV8RPT (+) and pV8RPT (-) Production and V8 Expression of protease derivatives
PG97S4DhCT [G] R6 used in this example is a plasmid that highly expresses a fusion protein of an E. coli β-galactosidase derivative and a human calcitonin precursor (hCT [G]), and the plasmid is plasmid pBR322 and plasmid pG97S4DhCT [G ] (See JP-A-5-328992 and FIG. 2). Escherichia coli W3110 containing the plasmid pG97S4DhCT [G] was deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology as Escherichai coli SBM323 on August 8, 1991, under the Budapest Treaty. No. 3503 (FERM BP-3503) has been granted.
[0053]
In order to express V8 protease gene derivatives I and II obtained by PCR, pG97S4DhCT [G] R6 was treated with XhoI and SalI, and a DNA fragment (3.1 kb) lacking the human calcitonin precursor gene part was subjected to agar gel electrophoresis. It was prepared by. This DNA fragment was ligated with the previously obtained V8 protease gene fragment with the XhoI and SalI sticky ends by T4 DNA ligase, transformed into JM101, and pV8RPT (+) in which V8 protease gene derivative I was cloned, PV8RPT (-) in which V8 protease gene derivative II was cloned was prepared (FIG. 3).
[0054]
The plasmid host strain used was JM101 strain (this strain can be obtained from Takara Shuzo Co., Ltd., In Vitrogen Catalog No. c660-00, etc.). The amino acid sequence of the fusion protein of V8 protease derivative and β-galactosidase derivative expressed from these plasmids is shown in FIG. After culturing JM101 / pV8RPT (+) and JM101 / pV8RPT (-) in 100 ml LB medium (0.5% yeast extract, 1.0% tryptone, 0.5% NaCl) at 37 ° C until OD660 reaches 1.0, then isopropylthiogalacto Pyranoside (IPTG) was added to a final concentration of 2 mM to induce expression. After the addition, the culture was further continued for 2 hours, and the cells were collected by centrifugation and suspended in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) so that OD660 was 5.
[0055]
This suspension was crushed with an ultrasonic crusher (Cellruptor; Toago Denki Co., Ltd.), the insoluble fraction was removed by centrifugation at 12000 rpm for 5 minutes, and the supernatant fraction was used as a crude enzyme solution. A synthetic substrate (Z-Phe-Leu-Glu-4-nitranilide; manufactured by Boehringer Mannheim) was used for measuring the activity of V8 protease. After mixing 940 μl of 100 mM Tris-HCl (pH 8.0) buffer with 20 μl of 10 mM Z-Phe-Leu-Glu-4-nitranilide solution (DMSO solution), add 40 μl of the crude enzyme solution for 5 minutes at room temperature. The increase in absorption at 405 nm due to the reaction was measured. Hitachi spectrophotometer U-3200 was used for the measurement.
[0056]
As a result, both JM101 / pV8RPT (+) and JM101 / pV8RPT (-) showed an enzyme activity of 8 μg / ml in the crude enzyme solution prepared from these cells, and the fusion protein with β-galactosidase derivative It was found active in the form and lacking the prepro sequence. Moreover, since activity was recognized also in pV8RPT (-) which deleted the C terminal repeat sequence, it turned out that this arrangement | sequence is not essential for activity.
[0057]
Since the production amounts of V8 protease derivatives I and II produced by JM101 / pV8RPT (-) and JM101 / pV8RPT (+) are low and their bands cannot be confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), purification Operation is difficult. Considering that the growth of bacterial cells stops with the addition of IPTG, and that the high-molecular-weight protein is decreased in the induced bacteria compared to the non-induced bacteria, the intracellularly expressed V8 protease derivative is It is considered that lethality is a cause of low expression.
[0058]
Example 3  Expression vector pV8D Making
In the case of V8 protease, since it has activity even when a β-galactosidase derivative is fused to the N-terminal side as described above, it cannot be inactivated by fusion only on the N-terminal side. Therefore, an attempt was made to inactivate V8 protease by further fusing a part of aminoglucoside 3'-phosphotransferase to the C-terminal side. For the C-terminal fusion, the EcoRV site before the repeat sequence was used.
[0059]
The plasmid pV8D related to the V8 protease derivative was prepared by the procedure shown in FIG. Prepare Bgl II-Sal I fragment (3.0 kb) and EcoRV-Bgl II fragment (0.7 kb) from pV8RPT (-) and ligate with Nar I-Sal I fragment (0.2 kb) prepared from pG97S4DhCT [G] R10 As a result, pV8hCT [G] was obtained. PG97S4DhCT [G] R10 can be prepared from plasmid pBR322 and plasmid pG97S4DhCT [G] in the same manner as pG97S4DhCT [G] R6 described above (see JP-A-5-328992 and FIG. 2).
[0060]
Next, the hCT [G] portion (0.1 kb BstE II-Sal I fragment) of the obtained pV8hCT [G] was pUC4K (Vieira, J. and Messing, J., Gene 19, 259 (1982), Pharmacia Ca. PV8D was prepared by replacing the 0.8 kb SmaI-SalI fragment containing the aminoglucoside 3′-phosphotransferase gene region of No.27-4958-01). The amino acid sequence of the V8 protease derivative fusion protein (V8D fusion protein) expressed from this plasmid is shown in FIG.
[0061]
This fusion protein has a structure in which a β-galactosidase derivative and a part of an aminoglucoside 3'-phosphotransferase are fused to the N-terminus and C-terminus of V8 protease via an R6 linker sequence, respectively. The R6 linker has the following sequence, that is, RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1), and has a structure in which the peptide bond at the center of RR in the sequence is cleaved by OmpT protease of E. coli.
[0062]
Example 4  V8D Fusion protein expression
E. coli JM101 (JM101 / pV8D) transformed with pV8D by a conventional method is cultured in 100 ml of LB medium at 37 ° C to an OD660 of 0.6, and IPTG is added to a final concentration of 2 mM to produce a V8D fusion protein. Induced. Culturing was continued for another 2 hours after the addition, and then the cells were collected by centrifugation. In the case of this strain, there was no phenomenon that cell growth stopped with the induction of fusion protein expression like JM101 / pV8RPT (+) and JM101 / pV8RPT (-), and no V8 protease activity was observed in the cell. .
[0063]
Moreover, it was observed by the microscope that the inclusion body was formed in the microbial cell. FIG. 7 shows the results of 16% SDS-PAGE of the insoluble fraction and the soluble fraction obtained by ultrasonically disrupting the cells before and after induction and the cells. It can be seen that the 60 kDa V8D fusion protein is highly expressed in the cells after induction, and that it is in the insoluble fraction because it forms an inclusion body.
[0064]
Since a part of the aminoglucoside 3′-phosphotransferase was fused to the C-terminal side of the V8 protease derivative, it became inactive without taking the original three-dimensional structure in the microbial cell, and the growth of the microbial cell was not inhibited. It was thought that it was highly expressed to the level of formation. In addition to the 60 kDa V8D fusion protein, a 27 kDa protein was also found in the insoluble fraction, and this protein was separated from the SDS PAGE gel and the amino acid sequence was examined. Degradation including the 282nd and subsequent methionine from the N-terminal of the V8D fusion protein. It turned out to be a thing.
[0065]
Example 5 FIG.  V8D Fusion protein OmpT Protease processing
The bacterial cells obtained by the culture shown in Example 4 were suspended in 10 ml of TE buffer, and sonicated to disrupt the bacterial cells. Thereafter, the inclusion body was recovered by centrifugation. The obtained inclusion body was again suspended in 10 ml of deionized water and then centrifuged to wash the inclusion body. After diluting the inclusion body with deionized water so that the OD660 value is 100, collect 150 μl, collect 25 μl of 1M Tris-HCl (pH 8.0), 2.5 μl of 1M dithiothreitol (DTT), and urea. 120 mg was added to dissolve the inclusion bodies, deionized water was added to 500 μl, and the mixture was heated at 37 ° C. for 2 hours.
[0066]
FIG. 8A shows the results of 16% SDS-PAGE before and after heating. It can be seen that bands having molecular weights of 12 kDa, 26 kDa, and 22 kDa, corresponding to parts of β-galactosidase derivative, V8 protease derivative, and aminoglucoside 3'-phosphotransferase, respectively, appear in the sample after heating.
On the other hand, FIG. 8B shows that the V8D fusion protein is expressed using the OmpT protease-deficient strain W3110 M25 (Sugimura, K. (1987) Biochem. Biophys. Res. Commun. 153, 753-759) as a host. The same operation was performed. In this case, since the above three bands cannot be detected during SDS-PAGE, the fusion protein is processed by OmpT protease (Sugimura, K. and Nishihara, T. (1988) J. Bacteriol., 170, 5625-5632). It is judged as specific cleavage by.
[0067]
Furthermore, when a 26 kDa band was cut out from SDS-PAGE and the N-terminal amino acid sequence was examined, it was confirmed that it was cleaved at the center of the RR part of the R6 sequence (RLYRRHHRWGRSGSPLRAHE) (SEQ ID NO: 1). It was confirmed that it was carried out specifically by OmpT protease. The above operation is carried out in the presence of 8M urea at the time of dissolution of inclusion bodies and 4M urea at the time of processing. As a result, OmpT protease is resistant to such high concentrations of urea and dissolves from inclusion bodies. It was shown for the first time that the fusion protein produced can be cleaved specifically.
[0068]
Example 6  Recombinant V8 protease (V8D) Refolding
The processed sample was diluted 20-fold with 0.4 M potassium phosphate buffer (pH 7.5) and left overnight on ice. By this operation, recombinant V8 protease (V8D) was refolded, and an activity corresponding to 30 μg / ml was obtained when measured according to the above-mentioned method. The refolding efficiency was about 20%. FIG. 9 shows the results of 16% SDS-PAGE before and after the refolding operation.
[0069]
When recombinant V8 protease (V8D) is refolded, β-galactosidase derivatives, aminoglucoside 3′-phosphotransferase-derived proteins, and other E. coli-derived proteins are degraded by acting as powerful proteases. The protein band disappears. Therefore, after the refolding operation, only the recombinant V8 protease (V8D) remains as the main protein in the sample, and a significant reduction in the subsequent purification operation can be expected.
The recombinant V8 protease (V8D) obtained in this way has a C-terminal deletion of 56 amino acids compared to the natural type in the construction of the gene, but the activity is maintained. It was found that it is not essential for activity.
[0070]
Example 7  Recombinant V8 protease obtained by refolding from inclusion bodies (V8D) Substrate specificity
In order to compare the substrate specificity of the recombinant V8 protease (V8D) obtained by refolding with that of the natural type, a fusion protein of human calcitonin precursor (hCT [G]) was used as the substrate, and both proteases were used for the protein. An experiment was conducted to release hCT [G] by the action of.
[0071]
The human calcitonin fusion protein used in the experiment has a structure in which a β-galactosidase derivative (108 amino acids) and hCT [G] are fused via a linker having glutamic acid, and the natural V8 protease is a carboxylate of this glutamic acid residue. The peptide bond on the side can be cleaved to release hCT [G]. An example of a plasmid having a gene encoding the fusion protein is pG97S4DhCT [G] R4 (see JP-A-5-328992).
[0072]
An amount equivalent to 1.2 μg of activity in terms of natural V8 protease in 1 ml of a 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 After adding the recombinant V8 protease (V8D) and reacting at 30 ° C for 1 hour, the reaction mixture was subjected to high-performance liquid chromatography (linear gradient using 0.1% trifluoroacetic acid (TFA) and 0.1% TFA / 50% acetonitrile). Elution).
[0073]
As the recombinant V8 protease (V8D), a sample after refolding was directly used, and a sample obtained by allowing a commercially available natural V8 protease to act on the same conditions was used as a comparative control.
FIG. 10 shows the elution pattern of high performance liquid chromatography. It was confirmed that the cleavage pattern for human calcitonin fusion protein was the same between recombinant V8 protease (V8D) and native V8 protease, and both proteases were equivalent in terms of substrate specificity.
[0074]
Example 8 FIG.  Examination of the C-terminal fusion site of V8 protease
In order to produce a fusion protein in which the C-terminal side of the V8 protease portion was extended by 2, 3, 4, 6 and 8 amino acids compared to the V8D fusion protein, the PCR primers shown in FIG. 11 were synthesized. PV8F encoding a fusion protein (V8F fusion protein) of the type extended by 3 amino acids was prepared by the following method. Using primer b and primer I shown in Fig. 1 (b), 0.1 Vg of pV8RPT (-) prepared in Example 1 as template DNA was used for amplification reaction on the V8 protease gene side, followed by EcoRI and SacI. A 0.1 kb gene fragment was prepared by cutting.
[0075]
On the other hand, after amplification reaction of R6 linker sequence and aminoglucoside 3 ′ phosphotransferase gene part using primer g and primer h and 0.1 μg of pV8D as template DNA, a 0.3 kb gene fragment was prepared by digestion with EcoT22I and SacI. . In addition, PCR followed the conditions of Example 1. The 0.1 kb and 0.3 kb genes thus obtained were ligated to the pV8D EcoRI-EcoT22I fragment (4.2 kb) to prepare pV8F (see FIGS. 12 and 14).
For primers a, c, d, e and primer g, pV8H, pV8A, pV8D2 and pV8Q were prepared by carrying out the same procedure (however, NdeI was used instead of SacI for the production of pV8H). The combinations of primers and template DNA used for the preparation of these plasmids are shown below.
[0076]
pV8H: Prepared from a PCR product obtained by combining primer a, primer I, pV8RPT (-) and primer f, primer h, pV8D.
pV8A: Prepared from a PCR product obtained by combining primer c, primer I, pV8RPT (-) and primer g, primer h, pV8D.
pV8D2: prepared from a PCR product obtained by combining primer d, primer I, pV8RPT (-) and primer g, primer h, pV8D.
pV8Q: prepared from a PCR product obtained by combining primer e, primer I, pV8RPT (-) and primer g, primer h, pV8D.
[0077]
These plasmids produced fusion proteins in which the C-terminal portion of the V8 protease region was extended by 2, 4, 6 and 8 amino acids, respectively, compared to the V8D fusion protein shown in Example 4 (FIG. 13, FIG. 13). 14).
FIG. 13 shows the results obtained by transforming these plasmids into the JM101 strain and examining the expression of each fusion protein by the same procedure as in Example 4. Inclusion body formation was observed in pV8H, pV8F and pV8D, and the resulting inclusion bodies could be refolded into active recombinant V8 protease by performing the procedure shown in Example 5. On the other hand, in the case of pV8A, pV8D2, and pV8Q, inclusion bodies were not obtained, and V8 protease activity was observed in the soluble fraction.
[0078]
In the case of these plasmids, the expressed fusion protein has protease activity, and inhibition of growth is considered to cause no inclusion body formation. In other words, in order to express V8 protease as an inactive fusion protein, it is important to fuse it before the 215th phenylalanine from the N-terminal. When fused beyond this site, the V8 protease part has its original three-dimensional structure. It was found that since a fusion protein formed and having protease activity was produced, growth was inhibited and high expression was impossible.
[0079]
【The invention's effect】
According to the present invention, a target polypeptide can be produced in a large amount at low cost. In particular, in the present invention, S. aureus V8 protease was examined as a target polypeptide. However, by using a safe host cell, for example, Escherichia coli, the high-purity enzyme can be produced in large quantities and at low cost using genetic recombination technology. Efficient purification / manufacturing has become possible.
[0080]
[Sequence Listing]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[Brief description of the drawings]
FIG. 1 shows Staphylococcus aureus (Staphylococcus  aureusFIG. 3 is a diagram showing the structure (a) of the V8 protease gene and the base sequence (b) of the PCR primer used for cloning of the gene of the present invention.
FIG. 2 is a diagram showing the production process of pG97S4DhCT [G] R6 and pG97S4DhCT [G] R10.
FIG. 3 is a diagram showing a process for preparing plasmids pV8RPT (+) and pV8RPT (−).
FIG. 4 shows the amino acid sequence of the fusion protein encoded in plasmids pV8RPT (+) and pV8RPT (−).
FIG. 5 is a diagram showing a process for preparing plasmid pV8D.
FIG. 6 shows the amino acid sequence of the fusion protein encoded in plasmid pV8D.
FIG. 7 is an electrophoretogram showing that the fusion protein of the present invention forms an inclusion body and moves to an insoluble fraction, and is a photograph replacing the drawing.
FIG. 8 is an electrophoretogram showing that a V8D fusion protein is cleaved by a host-derived protease ompT to release V8 protease, and is a photograph in place of a drawing.
FIG. 9 is an electrophoretogram showing refolding of V8 protease released by ompT protease, and is a photograph instead of a drawing.
FIG. 10 shows that a fusion protein containing a human calcitonin precursor was prepared by recombinant V8 protease (A) or S. cerevisiae obtained by the method of the present invention. It is the chart which compared the product at the time of cut | disconnecting with V8 protease (B) from Aureus.
FIG. 11 shows the nucleotide sequences of primers used in the preparation of plasmids (pV8H, pV8F, pV8A, pV8D2 and pV8Q) containing DNAs encoding various fusion proteins.
FIG. 12 is a diagram showing the process of preparing plasmids pV8H, pV8F, pV8A, pV8D2 and pV8Q.
FIG. 13 shows the C-terminal amino acid sequence of the V8 protease encoded in the plasmids pV8D, pV8H, pV8F, pV8A, pV8D2 and pV8Q, and the inclusion bodies by the expression products (fusion proteins) of each plasmid. It is the figure which displayed the presence or absence of formation.
FIG. 14 shows an example of the amino acid sequence of a target polypeptide produced by the method of the present invention.

Claims (28)

In the method for producing the target polypeptide,
(1) a fusion protein comprising a protected polypeptide and a polypeptide of interest [wherein the fusion protein is represented by formula (1) ALB or formula (2) ALBLC, A and C are protected polypeptides, B Is a target polypeptide, and L is a linker peptide having a site specifically recognized by E. coli endogenous OmpT protease].
(2) culturing the transformed E. coli to express the gene, thereby generating an insoluble fusion protein constituting an inclusion body;
(3) After culturing, Escherichia coli is disrupted to separate the inclusion body, and then the insoluble fusion protein constituting the inclusion body is solubilized with a denaturing agent; and (4) the inclusion body is separated from the fusion protein. The target polypeptide is excised by cleaving in the region of the linker peptide L with the disrupted Escherichia coli endogenous OmpT protease present in the inclusion body fraction by operation;
A method for producing a target polypeptide comprising the above.   The method of claim 1, wherein the cleavage occurs by reducing the concentration of the denaturant.   The method according to claim 1 or 2, wherein the denaturant that solubilizes the fusion protein is urea, guanidine hydrochloride, or a surfactant.   The method according to claim 3, wherein the denaturant that solubilizes the fusion protein is urea, and the concentration of the urea is 1 to 6M.   5. The linker peptide according to claim 2, wherein the linker peptide contains 1 to 2 basic amino acid pairs in the linker peptide, and the linker peptide is a peptide composed of 2 to 50 amino acid residues. the method of.   The method according to any one of claims 1 to 5, wherein the linker peptide comprises 1 to 2 basic amino acid pairs at the N-terminal part and / or the C-terminal part of the linker peptide. Wherein the linker peptide comprises the amino acid sequence RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1) comprising the method of claim 6.   The method according to any one of claims 1 to 7, wherein the protective polypeptide is an E. coli β-galactosidase-derived polypeptide and / or an aminoglycoside 3'-phosphotransferase-derived polypeptide derived from transposon 903.   The said protection polypeptide A is E. coli β-galactosidase-derived polypeptide, and protection polypeptide C is an aminoglycoside 3′-phosphotransferase-derived polypeptide derived from transposon 903. the method of.   The method according to any one of claims 1 to 9, further comprising a step of causing refolding so that the target polypeptide becomes active.   The target polypeptide cleaved by OmpT protease in the presence of a denaturing agent is refolded by lowering the concentration of the denaturing agent to obtain an active target polypeptide. The method described in 1.   The method according to any one of claims 1 to 11, wherein the target polypeptide consists of 20 to 800 amino acid residues.   The method according to any one of claims 1 to 12, wherein the target polypeptide is a physiologically active polypeptide.   The bioactive polypeptide is motilin, glucagon, corticosteroid, corticotropin releasing hormone, secretin, growth hormone, insulin, growth hormone secreting hormone, pazopressin, oxytocin, gastrin, glucagon-like peptide (GLP-1), glucagon -Like peptide (GLP-2), glucagon-like peptide (7-36 amide), cholecystokinin, vasoactive intestinal polypeptide (VIP), pituitary adenolate cyclase activation polypeptide ( Pituitary adenolate cyclase activating polypeptide), gastrin releasing hormone, galanin, thyroid stimulating hormone, luteinizing hormone releasing hormone, calcitonin, parathyroid hormone (PTH, PTH (1-34), PTH (1-84)), peptide, histidine, Isoleucine (peptide histidine isoleucine (PHI), neuropeptide Y (neuropeptide Y), peptide YY (peptide YY), pancreatic polypeptide, somatostatin, TGF-α, TGF-β, nerve growth factor, fibroblast growth 14. The method of claim 13, which is a factor, relaxin, prolactin, diuretic peptide, angiotensin, brain derived neurotrophic factor (BDNF), or KEX2 endoprotease.   15. A method according to claim 14, wherein the diuretic peptide is ANP, BNP or CNP.   14. The method according to claim 13, wherein the target polypeptide is an enzyme.   17. The method according to claim 16, wherein the enzyme is a proteolytic enzyme.   18. The method according to claim 17, wherein the proteolytic enzyme is KEX2 endoprotease or the protease derivative.   18. The method according to claim 17, wherein the proteolytic enzyme is Staphylococcus aureus V8 protease or the protease derivative. The Staphylococcus aureus (Staphylococcus aureus) V8 protease derivative is SEQ ID NO: 7 amino acid sequence from position 125 to Val to 336-position Asp, or SEQ ID NO: 22, the amino acid sequence set forth in 23 or 24 it is made derivative the method of claim 19. A method for producing a target polypeptide using a gene recombination technique, comprising the following steps:
(1) The fusion protein comprises a protective polypeptide, a target polypeptide and a linker peptide, and the protective polypeptide is a β-galactosidase-derived polypeptide derived from E. coli and / or an aminoglycoside 3′-phosphotransferase-derived polypeptide derived from transposon 903. An endogenous OmpT protease in which the target polypeptide is a Staphylococcus aureus V8 protease derivative and the linker peptide region between the protective polypeptide and the target polypeptide is present in the outer membrane of E. coli E. coli is transformed with an expression vector having a gene encoding the fusion protein, which is a linker peptide region having a site specifically recognized by
(2) By expressing the gene in E. coli, an insoluble fusion protein constituting the inclusion body is produced, and the Staphylococcus aureus V8 protease derivative is inactive in the inclusion body. In state
(3) After crushing the cells and separating the fusion protein, obtain a fraction containing the E. coli OmpT protease, which is an endogenous protease, and the fusion protein,
(4) solubilizing the fusion protein with a denaturant,
(5) After solubilization, reducing the concentration of the denaturant to a state where the activity of E. coli OmpT protease is expressed, and cleaving the linker peptide region with the protease to obtain the target polypeptide from the fusion protein;
A method for producing a polypeptide of interest comprising:   22. The method according to claim 21, wherein the denaturing agent that solubilizes the fusion protein is urea, guanidine hydrochloride or a surfactant.   23. The method according to claim 22, wherein the denaturant that solubilizes the fusion protein is urea, and the concentration of the urea is 1 to 8M. The urea concentration 4 M during the processing step by E. coli OmpT protease, the method according to any one of claims 21 to 23.   The linker peptide is a peptide comprising a dibasic amino acid pair at the N-terminus and C-terminus of the linker peptide, or in the linker peptide, and the linker peptide consists of 2 to 50 amino acid residues. The method of any one of -24.   26. The method according to any one of claims 21 to 25, wherein the linker peptide has the amino acid sequence RLYRRHHRWGRSGSPLRAHE (SEQ ID NO: 1). The target polypeptide, SEQ ID NO: 7 amino acid sequence from position 125 to Val to 336-position Asp, or SEQ ID NO: 22, 23 or Staphylococcus aureus comprising the amino acid sequence set forth in 24 (Staphylococcus aureus 27) The method according to any one of claims 21 to 26, which is any of V8 protease derivatives.   28. The method according to any one of claims 21 to 27, further comprising the step of causing refolding so that the polypeptide of interest is in an active form.

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