JP5370982B2 - Method for producing recombinant protease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for supplying a large quantity of Staphylococcus aureus V8 protease at a low cost. <P>SOLUTION: The invention provides a vector containing an expression cassette containing a promoter acting in E.coli and a nucleotide obtained by the in-frame linking of a nucleotide encoding a prepro sequence of a glutamic acid-specific protease derived from Staphylococcus epidermis or a variant prepro sequence obtained by 5-amino acid substitution of a prepro sequence of Staphylococcus aureus V8 protease with a nucleotide encoding mature protease, and expressing an inactivating soluble protease in E.coli, a transformant containing the expression vector, and a method for the production of an inactivating protease containing a step to culture the transformant, recover the supernatant of the cultured product and the purifying the protease from the recovered supernatant of the cultured product. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a recombinant protease, and more particularly to a method for supplying a protease such as V8 protease useful for proteome analysis using a gene recombination technique.

  Proteases are essential for intracellular and extracellular protein metabolism and also play an important role in intracellular signal transduction. In addition to their biological importance, various proteases are used as research tools in the field of protein chemistry. Recently, a protein database has been enriched by genome analysis, and it has become possible to perform proteomic analysis that identifies thousands of proteins at once by performing mass analysis of cleaved peptides. In proteome analysis, trypsin that specifically cleaves the peptide bond on the carboxyl side of arginine and lysine, a protease that is specific only for arginine, and Staphylococcus aureus that specifically cleaves the carboxyl side of acidic amino acids, particularly glutamic acid ) V8 protease (GluV8, EC 3.4.21.19) is widely used.

  Since GluV8 retains enzyme activity even in the presence of the surfactant sodium dodecyl sulfate (SDS), it can be used for proteins in polyacrylamide gels containing SDS. GluV8 is currently commercially available as a preparation purified from the culture supernatant of the producing bacterium S. aureus V8 strain, but the cost can be reduced if recombinant protease can be expressed in E. coli. Furthermore, genetic modification also makes it possible to produce enzyme molecules with higher activity and stability. In addition, the purified sample derived from the pathogenic bacterium S. aureus cannot be denied the possibility of contamination with various virulence factors, so if the recombinant sample can be expressed and purified, the scope of use of this enzyme Is expected to spread.

  In gene expression systems using E. coli, although some proteases such as caspases can be produced, it is generally considered difficult to express many proteases including GluV8. As a cause of this, even if the transcription and translation of the transgene proceed in E. coli, there is a possibility that the construction of the higher order structure may not be successful. In particular, when it has a disulfide bond, it is difficult to form a correct pair. On the other hand, when normal folding occurs, the activity of the expressed protease is considered to cause toxicity to the host.

  Hamada et al. (Non-Patent Document 1) expressed GluV8 in Escherichia coli as an insoluble molecule by introducing a foreign amino acid sequence on both the N-terminus and C-terminus of mature GluV8. In this expression method, the recombinant molecule is expressed as an inclusion body that is a precipitate, so that after purification, solubilization, cleavage of the additional amino acid sequence, regeneration, and subsequent purification of GluV8 are required (patented) Reference 1). The yield of active protease in the solubilization (denaturation) -regeneration operation is 20% of the purified sample and is not high. Therefore, establishment of a simpler and more efficient active protease expression method is meaningful.

  GluV8 is a trypsin family endopeptidase consisting of 336 amino acids (Non-patent Document 2). There is a 12-fold structure of Pro-Asn (or Asp) -Asn tripeptide at the C-terminal part (Non-Patent Document 3), but this sequence is not involved in enzyme activity or higher-order structure formation. (Non-Patent Document 1). In S. aureus, it is expressed as an inactive form having no protease activity, but Asn68-Val69 is cleaved by processing with aureolysin, a thermolysin family enzyme, and converted into a mature form consisting of 268 amino acids (Non-patent Document 4). ).

The present inventors previously purified a glutamic acid-specific protease (GluSE) derived from Staphylococcus epidermidis, which is a related enzyme of Staphylococcus aureus GluV8, and cloned its gene (Non-patent Document 5). The GluSE gene encoded 282 amino acids and the expected molecular weight was 30,809. The gene did not encode the C-terminal repeat sequence present in GluV8 (FIG. 1A).
JP-A-8-187094 Yabuta, M., et al., Appl. Microbiol. Biotech. 44, 118-125 (1995) Stennicke HR and Breddam K., Handbook of Proteolytic Enzymes.San Diego: Academic Press, 243-246 (1998) Carmona C. and Gray GL, Nucleic Acids Res. 15, 6757 (1987) Shaw, L. et al., Microbiology 150, 217-228 (2004) Ohara-Nemoto, et al., Microb. Pathog. 33, 33-41 (2002)

  The object of the present invention is to provide a cheap and large-scale supply means of useful proteases such as S. aureus V8 protease.

In the amino acid sequence of GluSE cloned by the present inventors (SEQ ID NO: 2), the N-terminal prepro sequence (SEQ ID NO: 8) was 66 residues, two amino acids less than the GluV8 prepro sequence (SEQ ID NO: 5). Furthermore, as a result of analysis of the N-terminal processing process of GluSE secreted by Staphylococcus epidermidis, the pre-sequence of GluSE (SEQ ID NO: 8) is Met1-Ala27 (SEQ ID NO: 6), and the pro-sequence is Lys28-Ser66 (sequence) No. 7) (Ohara-Nemoto et al., Preparing for submission). As a result of the processing of the N-terminal prepro sequence, GluSE becomes a mature molecule consisting of 216 amino acids. Comparing the amino acid homologies of GluSE and GluV8, excluding 2 residues of pro sequence and GluV8 unique to C-terminal, 37% and 20.7% of pre-sequence, pro-sequence and mature molecule region, respectively. 59.1%, the homology of the prosequence region was the lowest (FIG. 1B).
The present inventors succeeded in mass-producing inactive GluSE as a soluble protein in an E. coli expression system, and found that the prepro sequence of GluSE is not cleaved by E. coli protease. Furthermore, a method for purifying an active protease by in vitro processing of a prepro sequence of GluSE was established. Based on this finding, the present inventors succeeded in mass production and purification of GluV8 in an E. coli expression system, and completed the present invention.
Furthermore, as a result of diligent research to make use of the pre-pro sequence of GluV8 and to make use of the E. coli expression system developed by the present inventors, the present inventors have also replaced five amino acids in the pre-pro sequence of GluV8. The present inventors have succeeded in obtaining a protease in a high yield and completed the present invention.
That is, the present invention is as follows.

[1] A promoter operable in Escherichia coli, and an expression cassette containing a polynucleotide in which a nucleotide encoding a prepro sequence of a glutamate-specific protease derived from Staphylococcus epidermidis and a nucleotide encoding a mature protease are linked in frame A vector expressing an inactive soluble protease in E. coli.
[2] The above [1], wherein the prepro sequence is a peptide having the amino acid sequence of SEQ ID NO: 8, or a peptide comprising an amino acid sequence consisting of at least the tyrosine at position 64 to the serine at position 66 of SEQ ID NO: 8. Expression vector.
[3] The expression vector according to [1] or [2], wherein the mature protease is S. aureus V8 protease.
[4] A polynucleotide comprising a promoter operable in E. coli and a nucleotide encoding a mutant prepro sequence obtained by substituting 5 amino acids of the prepro sequence of S. aureus V8 protease and a nucleotide encoding a mature protease linked in frame. A vector for expressing an inactive soluble protease in E. coli, comprising an expression cassette.
[5] The expression vector according to [4] above, wherein the mutated prepro sequence is a peptide having the amino acid sequence of SEQ ID NO: 17.
[6] The expression vector according to [4] or [5], wherein the mature protease is S. aureus V8 protease.
[7] Any one of [1] to [6] above, wherein the expression cassette further comprises a tag-encoding nucleotide linked in frame so as to add a tag to the C-terminus of the mature protease. The expression vector described in 1.
[8] The expression vector according to any one of [1] to [7], wherein the promoter is a T5 promoter / lactose operator.
[9] The above [3] or [6], wherein the S. aureus V8 protease lacks a 12 amino acid repeat sequence of 3 amino acids consisting of C-terminal Pro-Asp-Asn or Pro-Asn-Asn. Expression vector.
[10] A transformant comprising the expression vector according to any one of [1] to [9].
[11] The following steps:
Culturing the transformant according to [10] above,
A method for producing an inactive protease, comprising: recovering a culture obtained in the culture step; and purifying a protease from the recovered culture.
[12] The following steps:
Culturing the transformant according to [10] above,
Collecting the culture obtained in the culturing step;
A method for producing an active protease, comprising a step of purifying a protease from a collected culture, and a step of cleaving the prepro sequence by enzymatic treatment of the purified protease.
[13] A vector for producing an insoluble soluble protein in Escherichia coli containing a nucleotide encoding a prepro sequence of a glutamic acid-specific protease derived from Staphylococcus epidermidis.
[14] The above [13], wherein the prepro sequence is a peptide having the amino acid sequence of SEQ ID NO: 8 or a peptide comprising an amino acid sequence consisting of at least the tyrosine at position 64 to the serine at position 66 of SEQ ID NO: 8. Vector.
[15] A vector for producing an insoluble soluble protein in Escherichia coli, which contains a nucleotide encoding a mutant prepro sequence obtained by substituting the prepro sequence of S. aureus V8 protease with 5 amino acids.
[16] The vector according to [15], wherein the mutated prepro sequence is a peptide having the amino acid sequence of SEQ ID NO: 17.

  The expression vector of the present invention contains an expression cassette that can link a GluSE prepro sequence or a GluV8 mutant prepro sequence that is not cleaved in an E. coli expression system to the N-terminus of the target protein (mature protease). Moreover, it is possible to produce a large amount of a soluble chimeric protein, preferably an exogenous inactive protease. According to the transformant of the present invention, since the expression vector is included, the target chimeric protein or recombinant protein can be produced in a form that can be easily solubilized instead of inclusion bodies during culture. According to the method for producing a protease using the transformant of the present invention, a large amount of inactive soluble protease can be easily obtained, and a large amount can be supplied as an enzyme for proteome analysis or the like. Furthermore, it was also clarified that the protease activity of GluV8 produced according to the present invention is higher than that of GluSE, and that GluV8 has a higher utility value.

  The present invention relates to an expression cassette comprising a promoter operable in E. coli, and a polynucleotide in which a nucleotide encoding a prepro sequence of a glutamate-specific protease derived from Staphylococcus epidermidis and a nucleotide encoding a mature protease are linked in frame. A vector for expressing an inactive soluble protease in E. coli is provided.

  The greatest feature of the expression cassette contained in the expression vector of the present invention is that it contains a nucleotide encoding a prepro sequence of a glutamic acid-specific protease derived from Staphylococcus epidermidis as a constituent element.

  The glutamate-specific protease derived from Staphylococcus epidermidis is a protease cloned by the present inventors (Ohara-Nemoto, et al., Microb. Pathog. 33, 33-41 (2002)), its base sequence and amino acid. The sequence (SEQ ID NO: 2) is registered in Genbank (AB096695) and is publicly known.

  The pre-pro sequence of GluSE refers to a peptide having a regulatory function that inactivates the activity of the protease while being linked to the N-terminus of the mature protease and activates the protease by being cleaved. Specifically, a peptide having the amino acid sequence shown in SEQ ID NO: 8 (amino acid sequence at positions 1 to 66 of SEQ ID NO: 2) can be mentioned. As long as the prepro sequence of GluSE has the regulatory function, substitution or deletion of one or several (preferably 1 to 5, more preferably 1 to 3) amino acids in the amino acid sequence of SEQ ID NO: 8 Or a peptide having a sequence into which a mutation such as insertion has been introduced. As an example of the mutation of the amino acid sequence of the prepro sequence, 3 amino acids of Gly-Gly-Ser were inserted between methionine at position 1 and lysine at position 2 as shown in SEQ ID NO: 8 as described in Examples. A prepro sequence is mentioned.

  In the present invention, a peptide consisting essentially of the 27 amino acid sequence described in SEQ ID NO: 6 (the amino acid sequence at positions 1 to 27 of SEQ ID NO: 2) is referred to as a GluSE pre-sequence. In the present invention, a peptide consisting essentially of the 39 amino acid sequence described in SEQ ID NO: 7 (amino acid sequence at positions 28 to 66 of SEQ ID NO: 2) is referred to as a GluSE pro-sequence.

It was confirmed that the following five types of sequences were similarly effective as truncated mutant prosequences having a function of inhibiting enzyme activity. (1) Ser33-Ser66, (2) Ile49-Ser66, (3) Ile56-Ser66, (4) Asn61-Ser66, (5) Tyr64-Ser66
Therefore, the prepro sequence may be a peptide having the amino acid sequence of SEQ ID NO: 8 from the viewpoint of having a function of inactivating the activity of the protease located on the C-terminal side in expression in E. coli. Or a peptide comprising an amino acid sequence consisting of at least tyrosine at position 64 to serine at position 66 of SEQ ID NO: 8.

  In the present invention, a promoter operable in E. coli and a nucleotide encoding a mutant prepro sequence obtained by substituting 5 amino acids of the S. aureus V8 protease prepro sequence and a nucleotide encoding a mature protease are linked in frame. Also provided is a vector for expressing an inactive soluble protease in E. coli containing an expression cassette comprising the polynucleotide.

  In the present invention, Staphylococcus aureus V8 protease (EC 3.4.21.19, Genbank accession number: Y00356, as an example of a mature form, a protein having an amino acid sequence at positions 69 to 336 of SEQ ID NO: 1, hereinafter abbreviated as “GluV8”. The prepro sequence in some cases) refers to a peptide having a regulatory function that inactivates the activity of the protease while being linked to the N-terminus of the mature protease, and activates the protease by being cleaved. Specific examples include a peptide having the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence at positions 1 to 68 in SEQ ID NO: 1). The prepro sequence of GluV8 has a pre-sequence at its N-terminal side (specifically, a peptide having the amino acid sequence described in SEQ ID NO: 3), and a pro-sequence at its C-terminal side (specifically, described in SEQ ID NO: 4). It is also possible to classify them as peptides having the amino acid sequence of

  The mutated prepro sequence of GluV8 is a peptide having a regulatory function that activates the protease by inactivating and cleaving the activity of the protease while being linked to the N-terminus of the mature protease, In this expression system, the amino acid sequence is substituted so that it is not cleaved. The mutated prepro sequence of GluV8 is a substitution of at least 4 amino acids, preferably a substitution of 5 amino acids, more preferably a pro sequence (amino acid sequence at positions 28 to 68 in SEQ ID NO: 5) in the amino acid sequence shown in SEQ ID NO: 5. It is a sequence in which substitution of 5 amino acids is introduced. As a preferable specific example of the 5-amino acid substitution, the mutated prepro sequence of GluV8 is a peptide having the amino acid sequence described in MKGKFLKVSSLFVATLTTATLVSSPAANALSSKAMHNHPQQTQSSKQQTPKIQKGGNLKPLQQRSHPS (SEQ ID NO: 17). As long as the GluV8 mutant prepro sequence has the above-mentioned function, the amino acid sequence of SEQ ID NO: 17 is further substituted with one or several amino acids (preferably 1 to 5, more preferably 1 to 3). It may be a peptide having a sequence into which a modification such as deletion or insertion has been introduced. Examples of further modification of the amino acid sequence of the mutated prepro sequence of GluV8 include those in which 3 amino acids derived from an expression vector are inserted between Met at position 1 and Lys at position 2 (see FIG. 6b). As another example of modification, in the peptide having the amino acid sequence shown in SEQ ID NO: 17, a 4-amino acid substitution mutant in which Pro at position 67 is replaced with Ala and position 67 is returned to the wild-type GluV8 amino acid. A prepro sequence is mentioned.

  In the present invention, the protease to be expressed is not only a protease suitable for protein analysis and preparation, but also any protease available in various industries, such as aminopeptidase, carboxypeptidase, dipeptidylpeptidase, signal peptidase, metal Endopeptidase.

  As a preferred protease, S. aureus V8 protease (EC 3.4.21.19, Genbank accession number: Y00356, as an example of a matured product, position 69 of SEQ ID NO: 1 has been difficult to produce as a recombinant protein so far. A protein having an amino acid sequence at position 336) and a glutamate specific protease from S. warneri (EC 3.4.21.9, Genbank accession number: AJ293885, Yokoi K et al., Gene, 281, 115-122 (2001), eg sequence) Protein having the amino acid sequence of No. 18), blood coagulation-fibrinolytic protease, Factor Xa (EC 3.4.21.6), plasmin (EC 3.4.21.7), tissue type plasminogen activator (t-PA, EC 3.4. 21.68) or urokinase (u-PA, EC 3.4.21.73), thrombin (EC 3.4.21.5), collagenase commonly used in cell culture (EC 3.4.24.3), and algi from periodontopathic bacteria Porphyromonas gingivalis Down specific protease (arginyl Jinji Pine (Rgp1, Rgp2)), such as lysine-specific protease (lysyl Jinji Pine (Kgp)) and the like. A nucleotide encoding such a protease can be obtained by a method known per se based on the disclosed base sequence or amino acid sequence.

  When GluV8 (a protein having an amino acid sequence of positions 69 to 336 of SEQ ID NO: 1 as an example of a mature form) is selected as a preferred protease, Pro-Asp-Asn or Pro-Asn-Asn on the C-terminal side of the protease The modified GluV8 lacking the repeating sequence of 3 amino acids consisting of An example of the modified GluV8 may be either a complete deletion or partial deletion of the repetitive sequence. The number of the repetitive sequences is 12 in the case of GluV8 having the amino acid sequence of positions 69 to 336 of SEQ ID NO: 1 (corresponding to the amino acid sequence of positions 289 to 324), and 12 in the case of all deletions. It has a defect in the repeated sequence, and in the case of a partial defect, it has a defect in any of 1 to 11 repeated sequences. Nucleotides encoding such modified proteases can be obtained by genetic recombination techniques used in the art.

  In order to facilitate the purification of the protease to be expressed, it is preferable to design the expression cassette so that a tag is added to the C-terminus of the protease. In this case, in the expression cassette, a tag coding nucleotide is further linked in frame to the 3 'side of the mature protease coding nucleotide.

  As the tag, a tag used for purification of a recombinant protein is used without limitation, and His tag, Myc tag, glutathione S transferase (GST), green fluorescent protein (GFP), FLAG, maltose binding protein (MBP) The His tag is preferred because the tag portion has the smallest molecular weight, can be purified even in a denatured state, and the cost for purification is low.

  The “promoter operable in E. coli” contained in the expression cassette is not particularly limited, and any promoter incorporated in a known E. coli expression vector can be used. Since the protease to be expressed of the present invention is expected to cause toxicity to host E. coli when expressed constantly, a promoter comprising a combination of a promoter and an operator / suppressor is preferable, and a lactose promoter / lactose suppressor is preferable. Take an example. Such a promoter can be cut out from a commercially available expression vector and used by a method known per se. In particular, pQE-60 (Qiagen) contains a strong T5 promoter followed by two lactose operators, a downstream multicloning site, and a His tag coding sequence, with a His tag at the C-terminus of the expression product. Since it is an E. coli expression vector that can be added, it can be suitably used for the construction of the expression vector of the present invention.

  The expression cassette is constructed so that the promoter and various nucleotides are linked in-frame. Such a linking method is known in the art, and a person skilled in the art can prepare an expression cassette according to a method known per se. If the expression cassette is linked in frame, additional codons may occur at the linking site.

  The expression vector of the present invention may be any backbone of a plasmid vector or a phage vector as long as it contains the expression cassette and functions in E. coli. From the viewpoint of ease of handling, a plasmid vector is preferable. An expression vector constructed using the pQE-60 is more preferred.

  The expression vector of the present invention may further contain a drug resistance gene so that screening of transformants is easy. Examples of drug resistance genes include, but are not limited to, ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, chloramphenicol resistance genes, erythromycin resistance genes, and the like.

  The transformant of the present invention includes the expression vector.

  As a host to be transformed, any Escherichia coli capable of expressing a foreign protein can be used, and specific examples include Y1090, BL21, M15, SG13009, TG1, and XL1-Blue.

  The transformant of the present invention can be obtained by transforming the host with the expression vector by a method known per se.

An inactive recombinant protease can be produced using the transformant thus obtained. Such a manufacturing method is characterized by including the following steps:
(1) culturing the transformant of the present invention,
(2) a step of recovering the culture obtained in the culture step, and (3) a step of purifying protease from the recovered culture.

(1) Step of culturing the transformant of the present invention In the method for culturing a transformant, a medium and culture conditions can be appropriately set according to the host from which the transformant is derived. For example, in the case of a transformant containing an expression vector having pQE-60 as a backbone, an LB medium containing an appropriate antibiotic and a bacterial solution shaken overnight at 37 ° C. are diluted 3-fold with the same medium to obtain a 600 nm Incubate vigorously at 37 ° C. until the absorbance reaches 0.5-0.7. Add β-isopropylgalactopyranoside (final concentration 0.2 mM) and incubate at 30 ° C. for 5 hours.

(2) Step of recovering the culture obtained in the culturing step The culture is recovered by centrifuging the culture solution of the culturing step. The centrifugation conditions are usually 4,000 × g and 10 minutes.

(3) Step of purifying protease from the collected culture The collected culture (cells) is frozen and thawed in a lysis buffer (eg, Tris buffer (pH about 8) containing lysozyme, leupeptin, etc.). E. coli is lysed and centrifuged to collect the supernatant. As a result, the inactive protease produced from the expression vector of the present invention is solubilized and present in the supernatant. The inactive protease in the supernatant can be isolated and purified by a method known per se using chromatography or the like commonly used for protein purification. When a tag is added to the C-terminus of an inactive protease, it is preferable because it can be easily isolated and purified by affinity chromatography for the tag. For example, a protease to which a His tag is added is isolated and purified by affinity chromatography using an anti-His tag antibody-binding resin or a nickel-binding resin.

  The inactive recombinant protease obtained in this manner is less likely to cause self-digestion and the like, and can be stored for a long time. If necessary, the active protease can be provided at the time of use by subjecting the inactive protease to the following steps. The present invention also provides a method for producing an active protease. This manufacturing method includes the following step (4) in addition to the steps (1) to (3).

(4) Step of cleaving the prepro sequence by enzymatic treatment of the purified protease In this step, the inactive protease obtained in the step (3) is treated using an enzyme capable of cleaving the prepro sequence. Examples of the enzyme to be used include thermolysin, chymotrypsin, trypsin and the like.
The conditions for cleaving the prepro sequence with thermolysin include adding thermolysin at a weight ratio of 50: 1 to the purified recombinant protein, and in 10 mM borate buffer (pH 8), 2 mM CaSO ₄ , 0.005% TritonX100. The reaction at 36 ° C. for 4 hours is exemplified.

  Since the present inventors have clarified the location of the prepro sequence of GluSE and that the sequence is not cleaved by proteases normally present in E. coli, vectors containing nucleotides encoding the prepro sequence of GluSE are inactive in E. coli. It can be used as a vector for producing type soluble protein.

  By substituting 5 amino acids of the prepro sequence of GluV8 with the corresponding amino acid of the prepro sequence of GluSE, the present inventors have clarified that the sequence is not cleaved by proteases normally present in E. coli. A vector containing a nucleotide encoding a prepro sequence can be used as a vector for producing an insoluble soluble protein in E. coli.

  Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited to the following examples.

Materials Expression vectors pQE60 and pREP4 and Hot Star PCR kit are Qiagen (Tokyo), Talon affinity gel is Clontech (Palo Alto, CA, USA), Thermolysin is Sigma (St. Louis, Mo., USA), KOD Plus DNA polymerase is Toyobo (Tokyo), DNA modifying enzyme is Nippon Gene (Tokyo), glutamic acid specific fluorescent peptide substrate (Z-Leu-Leu-Glu-MCA) and leupeptin are peptide research institute (Osaka), DEAE Sephacel Molecular weight markers for low molecular weight SDS electrophoresis were purchased from Amersham Biosciences (Piscataway, NJ, USA), and azocasein from Sigma. Oligoprimers were purchased from GeneNet (Fukuoka City). For other general reagents, special grades were used.

Example 1 Preparation of an expression plasmid for a glutamate-specific protease 1
(1) Cloning of S. epidermidis glutamate-specific protease gene
Using S. epidermidis ATCC 14990 genomic DNA as a template, the following primers:
5'-TAT GGATCC AAAAAGAGATTTTTATCTATATGTAC; (5'-primer, SEQ ID NO: 9) and
5'-ATT GGATCC CTGAATATTTATATCAGGTATATTG; (3'-primer, SEQ ID NO: 10)
PCR was performed using. The underlined portion in the primer sequence indicates the introduced BamHI site. SEQ ID NO: 10 was a 3′-primer excluding the stop codon so that a histidine tag was added to the C-terminus. PCR conditions were as follows.
After 94 ° C. for 2 minutes, 94 ° C., 20 seconds, 55 ° C., 20 seconds, 72 ° C., and 1 minute temperature circle were performed 30 times to perform extension reaction at 72 ° C. for 4 minutes.
The DNA fragment obtained by PCR was treated with BamHI and then ligated to pQE60 which had been cleaved with BamHI and dephosphorylated separately. Escherichia coli Y1090 [pREP4] was transformed with the obtained expression plasmid (pQE60-GluSE). It was confirmed by DNA sequencing that no base mutation occurred in the insertion sequence of pQE60-GluSE.

(2) Cloning of S. aureus glutamate-specific protease gene
Using S. aureus ATCC 35984 genomic DNA as a template, the region encoding the full-length GluV8 (336 amino acids), the following primers:
5'-ATG GGATCC AAAGGTAAATTTTTAAAAGTTAGTTCT; (5'-primer, SEQ ID NO: 11) and
5'-ATT GGATCC CTGAATATTTATATCAGGTATATTG; (3'-primer, SEQ ID NO: 12)
Was amplified by PCR. The underlined portion indicates the introduced BamHI site. PCR conditions were as follows.
After 94 ° C. for 2 minutes, 94 ° C., 20 seconds, 55 ° C., 20 seconds, 72 ° C., and 1 minute temperature circle were performed 30 times to perform extension reaction at 72 ° C. for 4 minutes.
A DNA fragment obtained by PCR was ligated to a plasmid by the same procedure as in (1) above to prepare an expression vector (pQE60-GluV8), and E. coli Y1090 [pREP4] was transformed.

(3) Construction of chimera (S. epidermidis and S. aureus) glutamate-specific protease expression plasmid In the state where pQE60-GluSE and pQE60-GluV8 obtained in (1) and (2) above coexist as templates, the following primers :
3'-primer for amplification of GluSE prepro region (5'-ACTTGGGTAACTTTTATTTTGACTTGGT: SEQ ID NO: 13)
PCR was performed using a 5′-primer for amplification of the mature region of GluV8 (5′-GTTATATTACCAAATAACGATCGTCACC: SEQ ID NO: 14). PCR conditions were as follows.
After 94 ° C. for 2 minutes, a temperature circle of 94 ° C., 20 seconds, 55 ° C., 20 seconds, 72 ° C. and 6 minutes was performed 35 times, and an extension reaction was performed at 72 ° C. for 4 minutes.
During this PCR, homologous recombination occurred in the vector sequence region, which is the common part of both templates, and a 5.5-kb linear plasmid encoding the chimeric molecule GluSE-GluV8 was amplified. Subsequently, phosphorylation and self-ligation of the 5 ′ end of the 5.5-kb fragment were performed to prepare a circular plasmid. E. coli Y1090 [pREP4] was transformed with the obtained circular plasmid pQE60-GluSE-GluV8. The nucleotide sequence of the obtained plasmid DNA was determined, and it was confirmed that the expected chimeric molecule expression plasmid was constructed.

(4) Construction of a chimeric glutamic acid-specific protease deletion mutant expression plasmid In order to investigate the function of the propeptide on the N-terminal side of the protease, a molecule in which the propeptide of the GluSE-GluV8 chimeric molecule was deleted stepwise An expression plasmid was constructed. A 5′-primer into which a BamHI site was introduced so as to start from Ile49, Ile56, Asn61, and Tyr64 of the propeptide region (Lys28-Ser66) of GluSE was prepared. As the 3′-primer, PCR was carried out using the GluV8 3′-primer (SEQ ID NO: 12) as described above and the GluSE-GluV8 chimeric molecule expression plasmid as a template. PCR conditions were as follows.
After 94 ° C. for 2 minutes, a temperature circle of 94 ° C. for 20 seconds, 55 ° C., 20 seconds, 72 ° C. and 6 minutes was performed 30 times, and an extension reaction was performed at 72 ° C. for 4 minutes.
The DNA fragment obtained by PCR was digested with BamHI in the same manner as in (1) above and introduced into the BamHI site of pQE60.

Example 2 Expression and Purification of Recombinant Glutamate Specific Protease 1
According to a previous report (Matsumoto et al., J. Biol. Chem. 277, 34959-34966 (2002)), the transformant obtained in Example 1 was cultured at 37 ° C. in LB medium containing 50 μg / ml ampicillin. After overnight shaking culture, the bacterial solution was diluted 3-fold with the same medium and cultured vigorously at 37 ° C. until the absorbance at 600 nm reached 0.5 to 0.7. 0.2 mM β-isopropylgalacto After inducing the expression of the recombinant protein in the presence of pyranoside at 30 ° C. for 5 hours, the cells were collected. Dissolve the cells by freeze-thawing in lysate buffer (20 mM Tris-HCl, pH 8, 0.1 M sodium chloride, 10 mM imidazole) containing 0.5 mg / ml lysozyme and 10 μg / ml leupeptin, then centrifuge and remove the supernatant Was recovered and used as a bacterial lysate. Recombinant protein was purified by Talon affinity gel chromatography using eluent (10% glycerol, 0.1 M imidazole, pH 8.0). The purified sample was stored at −80 ° C. until use.

Thermolysin treatment and measurement of protease activity To purified recombinant protein, 0 to 1 μg of thermolysin was added in 10 mM boric acid (pH 8), 2 mM CaSO ₄ , 0.005% TritonX100, and heated at 36 ° C. for 4 hours. Thereafter, a part of the sample was added to a protease activity measurement solution [50 mM Tris-HCl (pH 8), 5 mM EDTA, 20 μM Z-Leu-Leu-Glu-MCA] and kept at 25 ° C. 5 mM EDTA was added as a thermolysin inactivating agent. Two hours later, the fluorescence of the measurement light 460 nm was measured using the fluorescence spectrum photometer F-4000 (Hitachi) as the excitation light 380 nm. In the activity measurement using azocasein as a substrate, the recombinant protein (1 μg) was reacted with 50 mM Tris-HCl (pH 8), 5 mM EDTA, 2 mg / ml azocasein (75 μl) at 37 ° C. After the time, 25 μl of ice-cold trichloroacetic acid solution (15 w / v%) was added to stop the reaction. After centrifugation, an equal volume of 0.5 M NaOH was added to the supernatant, and the absorbance at 440 nm was measured.

Purification of Staphylococcus epidermidis secreted GluSE
S. epidermidis ATCC 14990 was cultured on a dialysis membrane using Todd Hewitt agar medium at 37 ° C. under aerobic conditions (Ohara-Nemoto et al., Microb. Pathog. 33, 33-41 (2002). )). After overnight culture, the extracellular fraction was collected, diluted with water to lower the ionic strength, and then added to DEAE Sephacel (1 × 6 cm) equilibrated with 20 mM Tris-HCl (pH 8). After washing with equilibration buffer, elution was performed with 20 mM Tris-HCl (pH 8) containing 0.1 M sodium chloride. The purity of the sample was 80% (see FIG. 4A). Stored at -80 ° C until use.

SDS polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was performed using a 12.5% polyacrylamide gel. The sample was heat denatured at 94 ° C. for 10 minutes in a sample buffer containing 1% SDS. After electrophoresis, Coomassie brilliant blue (CBB) staining was performed.

Zymography
Samples dissolved in sample buffer containing 1% SDS were separated by SDS-PAGE on a 12% polyacrylamide gel containing 1 mg / ml azocasein without heat treatment. After electrophoresis, wash twice with 2.5% TritonX100 for 20 minutes, then wash twice with 50 mM Tris-HCl (pH 8) and 30 mM NaCl for 20 minutes, then replace with fresh Tris-HCl solution. Incubated for 12-16 hours at ° C. Thereafter, the gel was stained with CBB.

N-terminal amino acid sequence Recombinant protein and its thermolysin cleavage molecule were separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad). The protein band on the membrane was stained with CBB, and the N-terminal sequence was determined using the Precise 49X cLC protein sequencer (ABI) (Nemoto T. et al., J. Biol. Chem. 272, 26179-26187 ( 1997)).

Protein quantification Bovine serum albumin was used as a standard and quantified by the bicinchoninic acid method (Pierce, Rockford, IL, USA).

Results E. coli expression of Staphylococcus epidermidis GluSE To minimize changes in the prepro sequence located in the N-terminal region of the molecule, the expression vector pQE60 with an affinity tag on the C-terminal side was used to An expression vector pQE60-GluSE encoding a molecule containing all of the sequence (Met1-Ala27), propeptide (Lys28-Ser66), and mature region (Val67-Gln282) was constructed (FIG. 1A). In the recombinant molecule, Gly-Gly-Ser is inserted between Met1-Lys2, and 8 amino acid residues (Gly-Ser-His-His-His-His-His-His: SEQ ID NO: 15) are added to the C-terminal Gln282. Has been. GluSE expressed in this system was recovered in the soluble fraction without aggregation and precipitation. Thereafter, it could be purified by one-step affinity column chromatography. 20 mg of purified preparation was obtained per liter of culture solution. SDS-PAGE revealed three types of bands of 29, 30, and 32 kDa (FIG. 2, left panel, lane 1). As a result of N-terminal sequence analysis, the N-terminus was Lys28 for the 32 kDa molecule, Ser33 and Ser37 for the 30 kDa molecule, and Ile49 for the 29 kDa molecule (Table 1 and FIG. 1B). In other words, the 32 kDa molecule is a molecule in which Ala27-Lys28 is cleaved by a signal peptidase from E. coli, and the 30 kDa molecule and 29 kDa molecule with Ser33 at the N-terminus are molecules generated by autolysis in Glu32-Ser33 and Asp48-Ile49. (Table 1). The substance of the His36-Ser37 cleaving enzyme that produces a 30 kDa molecule with N-terminal Ser37 is unknown at present, but it was assumed to be due to a protease produced by E. coli.

  In vivo, GluV8 from S. aureus, which represents the family enzyme to which GluSE belongs, has been reported to be matured by cleavage of Asn68-Val69 corresponding to Ser66-Val67 of GluSE by aureolysin belonging to the thermolysin family. (Shaw, L. et al., Microbiology 150, 217-228 (2004) and FIG. 1B). Thus, when the purified recombinant GluSE obtained in Example 2 was treated with various concentrations of thermolysin, a 28 kDa molecule appeared depending on the thermolysin concentration (FIG. 2, left panel). The N-terminus of the 28 kDa molecule is Val67 (Table 1), consistent with the N-terminus of the GluSE preparation purified from the previously reported culture supernatant of Staphylococcus epidermidis (Ohara-Nemoto et al., Supra). As a result of zymography, azocasein degrading activity was observed in the 28 kDa molecule (FIG. 2, right panel). As a control, measurement was carried out under conditions containing only the highest concentration of thermolysin in the present experimental conditions, but no azocasein degradation was observed (FIG. 2, right panel, lane 10). These results indicate that the 32kDa recombinant molecule that retains all of the propeptide and the 29 and 30kDa molecules that retain part of the propeptide are inactive, and the 28kDa mature molecule is obtained by hydrolysis between Ser66 and Val67 by thermolysin. It is concluded that will occur.

E. coli expression system of S. aureus GluV8 We also tried to express GluV8 using the same pQE60 expression system, but at the best, only 0.05 mg / liter of culture broth was obtained. Is not shown). The amino acid sequence homology of the GluSE and GluV8 pro-sequence regions is 20.7%, which is considerably lower than the 59.1% homology of the mature region (FIG. 1B). Therefore, we hypothesized that the low expression of recombinant GluV8 is due to the low homology GluV8 pro region. Therefore, an expression vector encoding a chimeric molecule of GluSE prepro sequence Met1-Ser66 and GluV8 Val69-Ala336 was constructed (FIG. 1A). As a result of expressing the chimeric molecule, all of the recombinant molecules were recovered in the soluble fraction, and the yield was almost the same as that of GluSE (18 mg / l). As a result of SDS-PAGE, the purified preparation contained a major band of 43 kDa and a minor band of 42 kDa having a smaller molecular weight (FIG. 3, left panel, lane 1). When this preparation was treated with thermolysin, molecular species of 42, 40, and 38 kDa were sequentially generated (lanes 3-9). Of these, the 40 kDa molecular species had major azocasein degrading activity (FIG. 3, right panel). As a result of N-terminal amino acid sequence analysis, the N-terminus of both the 40 kDa molecular species and the 38 kDa molecular species was Val69, which was identical to that of mature GluV8 (Table 1). From the above results, it was revealed that GluV8 can be expressed in a large amount as a chimeric molecule prepro-GluSE-mature GluV8, and that active mature GluV8 can be obtained by thermolysin treatment.

Comparison of GluSE and GluV8 protease activities
The recombinant GluV8 molecule is converted to the same amino acid sequence as that of natural GluV8 by thermolysin treatment, except for the addition of the C-terminal 8-residue amino acid. Therefore, we examined whether the properties of the recombinant preparations were the same as those of the enzymes derived from Staphylococcus epidermidis and Staphylococcus aureus.
SDS-PAGE was performed on 4 samples of GluSE purified from the culture supernatant of Staphylococcus epidermidis, commercially available GluV8 derived from Staphylococcus aureus, and mature recombinant GluSE and GluV8. Both Staphylococcus epidermidis and recombinant GluSE are 28 kDa (Fig. 4A), S. aureus GluV8 is 38 and 40 kDa in two bands, and recombinant GluV8 is inadequately cleaved by thermolysin in addition to these two bands Therefore, the remaining bands of 42 and 43 kDa were observed. As described above, there was almost no difference in the SDS-PAGE pattern between the natural product of each protease and the recombinant molecule expressed in E. coli and matured in vitro.
Next, the protease activity was compared and examined using fluorescent peptide substrates. Considering the purity of each preparation, the protease activity of recombinant GluSE is comparable to that of natural GluSE preparation, and the activity of recombinant GluV8 is lower than that of the natural product if thermolysin is not sufficiently cleaved (rec1). It was revealed that the sample treated with 3 times the amount of thermolysin (rec2) had an activity equal to or better than that of natural GluV8. From the above, it was concluded that there is virtually no difference in specific activity between natural and recombinant samples.

  Surprisingly, there was a two-digit difference in protease activity between GluSE and GluV8 (note that the scale in FIG. 4B differs between GluSE and GluV8). However, on the other hand, the results of zymography using azocasein (FIGS. 2 and 3) did not give the impression that there was a difference of two orders of activity, so the protease activity using azocasein as a substrate was also measured. As a result, although GluV8 still showed higher activity, the difference from GluSE was about twice, which was clearly different from the two-digit difference when a fluorescent peptide substrate was used (FIG. 4C).

Why GluV8 full-length molecules cannot be expressed in E. coli
What is the molecular mechanism that allows expression of full-length GluSE molecules and pre-pro-GluSE chimeric molecules despite the difficulty in expressing GluV8 full-length E. coli? We focused on amino acids within the prosequence. In GluSE, there is neither glutamate nor aspartic acid that can be recognized by GluSE in the vicinity of Ser66-Val67, which is the processing site for the mature form. On the other hand, in the pro sequence of GluV8, Glu62 and Glu65 are present in the vicinity of Asn68-Val69 which is a processing site (FIG. 1B, surrounded by a square). Therefore, if a small amount of active molecule is generated from a recombinant prepro-GluV8 molecule by an E. coli endogenous protease, self-cleavage of Glu62-Gln63 and Glu65-His66 bonds in the propeptide will occur. The resulting GluV8 Gln63-Ala336 and GluV8 His66-Ala336 molecules have only 6 or 3 extra residues on the N-terminal side of the mature molecule (Val69-Ala336), and thus can exert protease activity. As a result, an activation cascade reaction may occur. Therefore, whether or not a molecule having a propeptide partial sequence at Val69, which is the N-terminus of mature GluV8, actually has activity was examined below.

  In this example, based on the GluSE-GluV8 chimera molecule, which has higher activity and can be examined in detail, a molecule in which the pro region was sequentially deleted from the N-terminal side was prepared (FIG. 1A). First, the molecules that have actually observed Ile49 and Ile56 at the N-terminus were expressed. Furthermore, in GluV8, molecules having Asn61 and Tyr64 at the N-terminus corresponding to the GluSE sites corresponding to Gln63 and His66, which are Nln-terminal Gln63 and His66, which can be produced by the presence of Glu62 and Glu65 were prepared (FIG. 1A). E. coli expressing these recombinant molecules was viable. The SDS-PAGE pattern of the purified sample decreased in molecular weight depending on the degree of deletion, but after treatment with thermolysin, it became a double band of 38 and 40 kDa (FIG. 5A). The protease activity of the truncation (truncated mutation) molecule was also suppressed in the same manner as the full-length molecule, and the same level of activity appeared upon treatment with thermolysin (FIG. 5B). Since truncation molecules also cause Ser66-Val67 cleavage by thermolysin, and the mature protease exhibits the same level of protease activity as the full-length molecule, deletion of the prosequence affects the three-dimensional structure of the mature part, including the processing site. It was speculated that it would not. In other words, subtilisin (Subbian et al., J. Mol. Biol. 347, 367-383 (2005)) and thermolysin (O'Donohue and Beaumont, J. Biol. Chem. 271, 26477-26481 (1996)) Unlike the sequence, the GluSE prosequence did not appear to function as an intramolecular chaperone.

  In addition, recombinant molecules with some pro-sequence on the N-terminal side of Val69 are strongly inhibited in protease activity, but shorter molecules, especially those with very short pro-sequences starting from Asn61 and Tyr64, are not processed. But apparent protease activity was detected. However, its activity was low, about 0.05% of the mature molecule (FIG. 5B) (FIGS. 5B and C). It was proved that the GluSE-GluV8 molecule starting from the position corresponding to Glu62 and Glu65 of GluV8 retains the activity though it is very weak.

Discussion In this study, we have obtained some insights into the biochemical properties of GluSE and GluV8 and the construction of their E. coli expression system. First, it was found that the pro domain of GluSE has a strong inhibitory activity on protease activity. Due to this inhibitory activity, a large amount of soluble GluSE could be expressed in E. coli. Aureolysin, a processing enzyme for GluV8 and GluSE, is a thermolysin family protein that cleaves peptide bonds on the amino group side of hydrophobic amino acid residues. However, it was fortunate that such a specific enzyme was not present in E. coli. In addition, by linking the GluSE prepro region to the mature region of GluV8, GluV8 self-activation was avoided and GluV8 could also be expressed. Recombinant GluSE and GluV8 were able to prepare active molecules with the same sequence as the natural enzyme by in vitro limited digestion with thermolysin after purification. The specific activity of these recombinant molecules was the same as that of the original fungal molecules. It was also found that the specific activity of GluV8 is superior to that of GluSE, although the degree varies depending on the type of substrate used. As a result of the GluV8 full-length expression experiment, it is clear that expression of an exogenous protease as a soluble molecule in Escherichia coli is difficult when activation by self-digestion occurs even if E. coli expression is attempted as an inactive form. became.

  GluSE Asn61-GluV8 and GluSE Tyr64-GluV8 chimeric molecules cannot be expressed in Escherichia coli due to their toxicity, provided that the GluV8 full-length molecule is correct that the cleavage of Glu62-Gln63 and Glu65-His66 also results in activity. I expected. In fact, the chimeric truncated GluV8 molecules expressed from Asn61 and Tyr64 showed weak protease activity, but surprisingly, these truncation molecules were in contrast to the practically impossible expression of full-length GluV8 in E. coli. E. coli expression was possible. The present inventors speculate that addition of a tetrapeptide derived from a vector sequence to the N-terminal side of Asn61 or Tyr64 further reduces the activity of the molecule starting with Asn61 or Tyr64. That is, 7 amino acid residues as a pro-sequence (met-gly-gly-ser-Tyr-Pro-Ser: SEQ ID NO: 16, lowercased foreign tetrapeptide) almost completely (99.95%) inhibited GluV8 protease activity However (FIG. 5), if the GluV8 full-length molecule is cleaved with Glu65-His66, the remaining pro region is only 3 residues, and the activity suppression is considered to be incomplete. Therefore, it is considered that E. coli expressing the full-length molecule of GluV8 cannot grow.

  Prasad et al. (Prasad, L. et al., Acta Crysta. 60, 256-259 (2004)) found that the α-amino group of Val69 at the N-terminus of GluV8 and the glutamic acid residue of the substrate peptide were obtained from the results of X-ray crystallography. It was proposed that the ionic bond with γ-carboxyl group determines the substrate specificity of GluV8. In fact, the GluSE-GluV8 chimeric molecule lacking most of the propeptides with Asn61 or Tyr64 at the N-terminus is slightly active, whereas all propeptides or molecules starting with Ile49 or Ile56 are active. The fact that the N-terminal α-amino group has moved from the original position of Val69 can also be explained by the fact that the activity decreases. The low homology between the GluV8 and GluSE pro-sequences can also be explained if the main role required for the pro-sequence is only to inhibit the appearance of the α-amino group at the position of Val69.

  In the past, proteases of infectious bacteria have been considered as exacerbations of infection. Today, it is estimated that nearly 15% of nosocomial infections worldwide occur due to S. aureus. On the other hand, Staphylococcus epidermidis is a resident bacterium. The difference in the activity of glutamate-specific proteases between the two bacterial strains revealed this time may explain the pathogenicity of Staphylococcus aureus. In the future, it is necessary to clarify the relationship between the protease activity and pathogenicity of Staphylococcus aureus.

  Many commercial proteins are already produced by recombinant technology. For example, most restriction enzymes are E. coli expression standards. Many soluble proteases are monomers and have no sugar chain modification. However, protease expression by the E. coli expression system was often expressed as inclusion bodies in order to avoid toxicity of protease activity. As a result, multi-step preparation such as solubilization, regeneration, and purification of active enzyme by chromatography was necessary. In particular, the denaturation, solubilization and regeneration steps with urea etc. take time, are difficult to automate, and the recovery rate tends to vary from experiment to experiment. On the other hand, in the expression methods of the examples, the inactive protease is recovered in the soluble fraction, so that purification is easy. One day each for expression and purification is complete. Based on the results so far, a purified preparation of about 20 mg per liter culture can be obtained, and then mature molecules can be obtained with an efficiency of almost 100%. The yield can be further increased by examining the culture conditions of E. coli and the induction conditions of recombinant molecules. Among the two types of proteases expressed this time, GluV8 is considered to have higher utility value than GluSE due to the past achievements and high specific activity.

  Depending on the type of protease, self-digestion may occur during storage. Therefore, the advantage of this method is that the protease can be stored in an inactive form for a long time and can be easily converted to the active form by thermolysin treatment at the time of use. Can be mentioned. Thermolysin was irreversibly deactivated by millimolar EDTA, so even if it was present in a small amount under the present experimental conditions, there was no problem. If it becomes a problem, it is possible to remove thermolysin after activation by affinity chromatography for C-terminal tag of 28 kDa GluSE or 40 kDa GluV8 which is the main band.

  The strategy of expressing E. coli as an inactive molecule by suppressing the autolytic reaction may be further applicable to the expression of other proteases. The periodontopathic bacterium Porphyromonas gingivalis expresses arginine-specific proteases arginyl ginsine pine (Rgp1 and Rgp2) and lysine-specific protease lysyl gin dipine (Kgp). Activation of Rgp1 and Rgp2 is caused by cleavage of Arg-Tyr bond, and activation of Kgp is caused by cleavage of Arg-Asp. In other words, even if Rgp1 or Rgp2 full-length molecules are expressed in E. coli, mature molecules will be produced in cascade by autolysis. Even in the case of Kgp, although the activation site is Arg-Asp, Arg and Lys may be cleaved because they are the same basic amino acid. In addition, in Kgp, Lys is located 5 residues from the processing site, so cleavage at this position will also cause activation. Although Rgp1 has already been reported for expression in E. coli, recombinant Rgp1 has also been obtained as an inclusion body (Margetts et al., Protein Expr. Purif. 18, 262-268 (2000)). The results of the Examples show that even gingipain may be expressed if the cleavage site undergoing self-processing is appropriately mutated. Substituting glutamic acid for the basic amino acid at the processing site is thought to stabilize the inactive form and to produce an active molecule by in vitro processing with GluV8.

Example 3 Preparation of Expression Plasmid for Glutamic Acid Specific Protease 2
All pQE60 (Qiagen) was used as an expression vector. Based on the expression plasmids of GluSE (wild type), GluV8 (wild type) and GluSE-V8 (a chimeric molecule of GluSE prepropeptide and GluV8 mature sequence) prepared in Example 1, an in vitro point mutagenesis method was used. Amino acid mutations were introduced. GluV8 (336 amino acids) has a 12 amino acid repeat structure of 3 amino acids (Pro-Asp-Asn or Pro-Asn-Asn) at the C-terminus, but a deletion of 52 amino acids including this was introduced (Table 2). As a result, the amino acid of GluV8 consisted of 284 amino acids, and the mature sequence portion (amino acids 69-284) was the same length as GluSE (amino acids 67-282). Amino acid substitutions were introduced at 2, 4 or 5 positions in the GluV8 propeptide (amino acids 1-68) by in vitro gene mutation and named GluV8mut2, mut4 and mut5, respectively. The amino acid to be introduced was matched with the amino acid of GluSE using the alignment results of the GluV8 and GluSE prosequences (Table 2 and FIG. 6b). Based on the report of Yokoi et al. (2001) regarding the amplification of Staphylococcus warneri glutamate-specific protease (named GluSW), a primer set with a BamHI site (underlined):
5 ′ primer, 5′-TTT GGATCC AAAGTTAAGTTTTTTACAGCA (SEQ ID NO: 19) and
3 ′ primer, 5′-GGC GGATCC CGCTGCGTCTGGATTATCGTA (SEQ ID NO: 20)
The DNA fragment encoding Lys2-Ala316 was amplified and inserted into the BamHI site of pQE60. A chimeric molecule of GluV8mut5 prepro sequence and GluSW mature sequence was prepared by PCR method.

Example 4 Expression and Purification of Recombinant Glutamate Specific Protease 2
In the same manner as in Example 2, the transformant containing the expression plasmid prepared in Example 3 was cultured. After induction of expression of the recombinant protein, the transformant was recovered and purified. The purified sample was stored at −80 ° C. until use.

Thermolysin treatment and measurement of protease activity In the same manner as in Example 2, the purified recombinant protein was treated with thermolysin and the protease activity was measured.

SDS polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was performed in the same manner as in Example 2 except that 11% polyacrylamide gel was used instead of 12.5% gel.

N-terminal amino acid sequence In the same manner as in Example 2, the N-terminal amino acid sequence of the recombinant protein and its thermolysin cleavage molecule was determined.

Protein quantification Protein was quantified in the same manner as in Example 2.

result
Expression of three glutamate-specific proteases and their derivatives
Yokoi et al. (Yokoi K., et al., Gene, 281, 115-122 (2001)) report purification and gene cloning of a glutamic acid specific protease GluSW of S. warneri M strain. GluSW (SEQ ID NO: 18) is not a typical repeat of 3 amino acids (Pro-Asp / Asn-Asn), but has a C-terminal sequence consisting of 34 amino acids including 11 residues of Asp and 16 residues of Asn. . C-terminal repeats are not present in GluSE. Except for the GluV8 C-terminal repeat structure and the GluSW Asp / Asn-rich sequence, the amino acid sequences of GluV8 Ala284 and GluSW Ser282 corresponding to Glu282 at the GluSE C-terminal were compared (FIG. 6). In the mature region, GluV8 and GluSW had the highest homology at 80.1%, followed by GluSE and GluSW at 61.1%, and GluV8 and GluSE at 58.5%, with slightly higher homology between GluSW and GluSE. The trend was similar for the pre and pro sequences, and both GluV8 and GluSW were more homologous than GluSE and GluV8 or GluSE and GluSW. In the three regions of pre, pro, and mature sequences, the mature portion had a higher homology of 58.8-80.1% than the other two regions, and the pro sequence had the lowest homology of 15.4-51.3%. From these, it can be seen that GluV8 and GluSW are more closely related. Secondly, there is a difference in the mutation rate of each region, which means that the structure required for the pro region to exert its function is necessary for the mature region to function (ie, exhibit enzymatic activity). It shows that there is a large degree of freedom of amino acid substitution compared to a simple structure.

Three glutamic acid-specific protease GluSE, GluV8 or GluSW genes encoding full-length molecules at the BamHI site of the pQE60 expression vector (Ohara-Nemoto et al., Microb. Pathog. 33, 33-41 (2002); Carmona and Gray, Nucleic Acids Res. 15, 6757 (1987); Yokoi et al., Gene, 281, 115-122 (2001)) or a derivative thereof was inserted to attempt E. coli expression. Gly-Gly-Ser derived from a restriction enzyme site is added to the N terminus of the expressed recombinant molecule, while His ₆ tag is added to the C terminus. Furthermore, based on 3 types of expression vectors, 9 types of vectors expressing their derivatives, 12 types in total, were expressed (Table 2).

Bacterial lysates containing 12 kinds of expressed molecules were separated by SDS-PAGE and stained with protein (FIG. 7). In lanes 1, 7, 8, 9, 10, and 12, a band of the expressed protein was confirmed (FIG. 7a, arrowhead). As will be seen later, a 44 kDa molecule was also expressed quantitatively in lanes 5 and 6, but because of the protein derived from E. coli at that position, expression could not be confirmed by lysate. When the separated protein was detected with a His _6- tag specific monoclonal antibody, the presence of a positive band was confirmed in all lanes (FIG. 7b). In many lanes, multiple bands were detected and found to be partially decomposed. Since the epitope His ₆ that reacts with this antibody is localized at the C-terminal, it was presumed that it was cleaved at a different part on the N-terminal side.

  The recombinant protein was purified from the bacterial lysate by affinity chromatography (Talon affinity gel) (FIG. 7c). The band intensity was considered to reflect the expression level. The expression level of GluSE (lane 1) was significantly higher than that of GluV8 (lane 2). In Example 2, the expression efficiency of GluV8 was increased by replacing the prepro sequence of GluV8 with that of GluSE (lane 3). This improvement was thought to be due to the replacement of the prepro sequence of GluV8 with the prepro sequence of GluSE, which is difficult to self-digest. However, as shown in FIG. 7a, as with GluV8, GluSE also produced a plurality of bands and partial decomposition occurred.

  When the prosequences of the two proteases are compared, in GluV8, there are amino acids that form a peptide bond that is most susceptible to digestion by GluV8, Glu62 and Glu65, near the C-terminus of the prosequence portion. On the other hand, in the GluSE prosequence, Glu40 is closest to the N-terminus of the mature sequence, and Asp that can be cleaved inefficiently is also closest to the C-terminus, Asp46. The present inventors have shown that GluV8 cleaved at the positions of Glu62 and Glu65 has an activity of about 1 / 1,000 of mature type (Nemoto et al., FEBS J. 275, 573-587 (2008)). . In other words, the low expression of GluV8 was presumed to be due to self-digestion in the host and damage to the host.

  In order to suppress self-digestion within the pro-sequence, an improved method of expressing using the original pro sequence of GluV8 was examined. The band intensity gradually increased as the amino acid sequence was changed to 2 amino acids (FIG. 7c, lane 4), 4 amino acids (lane 5), 5 amino acids (lane 6), and GluSE pro-sequence amino acids. In particular, all of the GluV8 prepro sequences (two Glu and one Asp) and the molecule in which two amino acids at the C-terminal end of the pro sequence were replaced with GluSE amino acids had the strongest band intensity (lane 6). .

  GluV8 has a 12-repeat structure of 3 amino acids (Pro-Asp-Asn or Pro-Asn-Asn) at the C-terminus (Carmona and Gray, Nucleic Acids Res. 15, 6757 (1987)). However, this sequence is not conserved in GluV8 family proteins. For example, since GluSE does not have this sequence, it is considered to be unrelated to protease activity. Molecules that are active even when GluV8 in which this portion is actually deleted are expressed have been obtained (Yabuta et al., Appl. Microbiol. Biotech. 44, 118-125 (1995)). Therefore, when the deletion molecule (GluV8mut5 ΔC) of 52 amino acids (amino acids 285-336) containing the C-terminal repeat sequence was expressed, the band intensity increased more than the non-deletion molecule (GluV8mut5, lane 6) (lane 7). ).

Comparison of constitutively inactive GluV8 and latent GluV8 expression efficiency Replacing the GluV8 prosequence with a self-digesting resistant amino acid as described above improves the yield of GluV8, and GluV8mut5 ΔC shows the highest expression. It was. However, it is unclear whether autolysis is still occurring during expression under these conditions. Therefore, we tried to express constitutively inactive GluSE and GluV8 as a control. Glutamate-specific protease family members are serine proteases, and GluSE Ser235 and GluV8 Ser237 are thought to be essential for active expression (Ohara-Nemoto et al., Microb. Pathog. 33, 33-41 (2002); Prasad Acta Crysta. 60, 256-259 (2004)). In fact, when Ser237 of GluV8 was replaced with Ala, no activity was shown by subsequent activation in vitro by thermolysin (Nemoto et al., FEBS J. 275, 573-587 (2008)). Thus, expression of the constitutively inactive molecules GluSE Ser235Ala, GluV8mut4 Ser237Ala, and GluV8mut5ΔC Ser237Ala that did not cause autolysis at all was achieved (FIG. 7, lanes 8-10). By the way, in the latent molecules GluSE already described (FIG. 7, lane 1), GluV8mut5 (lane 6) and GluV8mut5 ΔC (lane 7), if autolysis is completely suppressed, each homeostasis is inactive. The same level of expression as the type molecule is expected. In fact, when comparing GluSE (lane 1) with GluSE Ser235Ala (lane 8), GluV8mut5 (lane 6) and GluV8mut4 Ser237Ala (lane 9), GluV8mut5 ΔC (lane 7) and GluV8mut4 ΔC (lane 10) Expression was achieved.

Expression of S. warneri glutamate-specific protease (GluSW) and its chimeric molecule (GluV8-SW) Until now, there has been no report on the expression of GluSW in Escherichia coli and other hosts. Yokoi et al. (Gene, 281, 115-122 (2001)) purified GluSW produced by S. warneri, and the mature N-terminal sequence was GluV8 (Val67-Ile-Leu-Pro ---) or Unlike GluSE (Val67-Ile-Leu-Pro ---), it is reported that it was Arg64-Ala-Asn-Val-Ile-Leu-Pro ---. If so, the activation mechanism of Glu63-Arg64 by autolysis may be considered. Therefore, an E. coli expression system is constructed to clarify whether the expression method of this study can be applied to GluSW and whether subsequent in vitro activation occurs in GluSW by autolysis or by thermolysin family enzymes. It was decided.

  In wild-type GluSW, immunoblotting and purified samples showed three bands with a major band of 33 kDa, but the yield was quite low, and expression was not confirmed when the lysate protein was stained with CBB. (FIG. 7, lane 11). Within the pro-sequence, there are Glu28, Asp34, Asp51, Glu60, Glu63 and amino acids that are targets for autolysis (FIG. 6b). In particular, Glu60 and Glu63 near the pro-sequence C-terminal were also common to GluV8. In other words, as with GluV8, low GluSW expression was presumed to be due to self-digestion in the expression host.

  Mutation of 7 amino acids (3Glu, 2Asp and Ala-Asn at the end of the pro-sequence, Fig. 6b) of the GluSW pro-sequence, which was successful in GluV8, was thought to suppress the activation of GluSW by autolysis. . However, considering the high homology between the GluSW and GluV8 pro-sequences shown in Fig. 6a, rather than the complicated method of mutating seven types of amino acids, the previously created mutant GluV8 (GluV8 mut5) pro We thought that this purpose could be achieved by using the sequence to create a chimeric molecule (GluV8 mut5-SW). Thus, when a chimeric molecule was actually created and expressed, it was expressed as a single molecule of approximately 38 kDa (FIG. 7c, lane 12), and the expression level was improved until a band was observed at the lysate protein stage. (Figure 7a, lane 12).

Mass expression and purification of GluV8 and GluSW
The yield of GluV8mut5 ΔC was improved in the expression system in a small amount (12 ml) compared to the wild type molecule and the chimeric GluV8 of Example 2 (FIG. 7). It was shown that the expression level was greatly improved (FIG. 8a). 20 mg of purified preparation per liter culture was obtained. GluSW gave exactly 25 mg of sample per liter in exactly the same way.

In Vitro Processing of Expressed Recombinant Proteases Since the goal was to obtain large quantities of active proteases, we tested whether purified pro-type proteases could be converted to mature forms in vitro. Heating with purified GluSE (10 μg) or equimolar amounts of other recombinant proteases at 0.1 or 0.3 μg of thermolysin produces an intermediate molecular weight band with 0.1 μg treatment [recombinant protease / thermolysin (molar ratio) = 100/1]. Although produced, those treated with 0.3 μg (molar ratio, 33/1) gave almost a single band (FIG. 9a). GluV8mut5 (lane 6) produced two bands, but as shown in FIG. 3, GluV8 produced by the original S. aureus produces two bands at the same position. It was thought that the molecule was cleaved within the C-terminal repeating structure.

  The activity of three glutamate-specific proteases was measured using Z-Leu-Leu-Glu-MCA peptide as a substrate. The purified sample showed no activity, but the activity was expressed by treatment with thermolysin. The specific activity of GluV8 was overwhelmingly higher than the other two species. GluSW was comparable to GluSE (Figure 9b). FIG. 9c shows the same data re-plotted with the focus on GluSE and GluSW, except for the highly active GluV8. In both of the two thermolysin treatment conditions, 43% (0.1 μg thermolysin treatment) and 86% (0.3 μg treatment) GluSW were more active than GluSE. The large difference in specific activity of GluV8 and GluSE with respect to Z-Leu-Leu-Glu-MCA is not due to artificial mutations caused by genetic manipulation, but the differences in the activities of the enzymes produced by the respective bacteria. It was reflected as it was.

  Further investigation was made on such a large difference in specific activity. The activity was measured using azocasein as a substrate. The order of specific activity was similar to that of GluV8> GluSW> GluSE, but unlike Z-Leu-Leu-Glu-MCA, There was no significant difference between the two proteases. In addition, a comparison using these two types of substrates revealed that there was no difference in activity even in molecules lacking the 52 amino acids at the C-terminus of GluV8.

N-terminal sequence of expressed GluSW When the N-terminal sequence of expressed recombinant GluSW was determined, GluV8mut5-SW had Leu30 of GluV8 (Table 3). In other words, the GluV8mut5-SW chimeric molecule was cut between the original pre-pro sequence and the pro sequence in the E. coli expression system. On the other hand, the N-terminus of wild-type GluSW was Arg64, lacking most of the pro-sequence and retaining only the last 3 amino acids of the pro-sequence. The Glu63-Arg64 bond is a bond that can be cleaved by GluSW, and it was thought that autolysis occurred as expected. Therefore, it was confirmed in GluSW that the yield of expression was improved by suppressing autolysis.

FIG. 1 shows a comparison of the amino acid sequences of GluSE and GluV8 and an overview of the expressed molecules. (A) Schematic representation of Staphylococcus epidermidis GluSE, Staphylococcus aureus GluV8, GluSE-GluV8 chimeric molecules and N-terminal deleted molecules expressed in the examples. (B) The amino acid sequences of the N-terminal regions of GluSE, GluV8 and GluSE-GluV8 chimeric molecules were compared. Amino acids are indicated by one alphabet. Amino acids common to GluSE and GluV8 are underlined. A two residue deletion (hyphen) was introduced in GluSE to maximize homology. The arrow indicates the cleavage site of the recombinant purified preparation and the band (FIGS. 2 and 3) detected by the thermolysin treatment. The downward arrow is the cleavage site of GluSE, and the upward arrow is the cleavage site obtained with the GluSE-GluV8 chimeric molecule. Glu62 and Glu65 of GluV8 noted in this example are surrounded by a square. FIG. 2 shows the expression of recombinant GluSE. Purified recombinant GluSE (0.8 mg / ml, 80 μl) was incubated at 0 ° C. or 36 ° C. and in the absence or presence of thermolysin. After 4 hours (left panel), 1% SDS was added to a part (1.5 μg) and heat-denatured, followed by SDS-PAGE and CBB staining. (Right panel) After adding 1% SDS to a thermolysin-treated sample (6 μg), it was separated by SDS-PAGE without heat treatment. Thereafter, zymography was performed. Stored at 0 ° C in the absence of thermolysin (lane 1); stored at 36 ° C (lane 2); or at 36 ° C thermolysin 0.3 ng (lane 3), 1 ng (lane 4), 3 ng (lane 5), 10 ng ( Lane 6), 30 ng (lane 7), 0.1 μg (lane 8), 0.3 μg (lane 9). Lane 10: A control similarly treated with a solution containing only the same amount (0.3 μg) of thermolysin as Lane 9. Lane 11: Staphylococcus epidermidis culture supernatant protein (1.5 μg). Lane 12: Equal mixture of lanes 1 and 11. M: Molecular weight marker. FIG. 3 shows the processing of the purified chimeric molecule GluSE-GluV8 by thermolysin. Purified recombinant GluSE-GluV8 chimeric molecule (6.5 μg / 80 μl) was incubated for 4 hours at 0 ° C. or 36 ° C. and in the absence or presence of thermolysin. (Left panel) Subsequently, SDS was added to a part (0.5 μg), heat-denatured, and then separated by SDS-PAGE. (Right panel) After adding SDS to a part (0.5 μg), it was separated by SDS-PAGE without heat treatment. Thereafter, zymography was performed according to the procedure shown in the examples. Stored at 0 ° C in the absence of thermolysin (lane 1); stored at 36 ° C (lane 2); or at 36 ° C 1 ng (lane 3), 3 ng (lane 4), 10 ng (lane 5), 30 ng ( Lane 6), 0.1 μg (lane 7), 0.3 μg (lane 8), 1 μg (lane 9); a control containing the same amount (1 μg) of thermolysin as lane 9 was similarly treated (lane 10). M represents a molecular weight marker. FIG. 4 shows a comparison of staphylococcal proteases and recombinant proteases. (A) 0.5 μg each of GluSE (native GluSE) purified from Staphylococcus epidermidis, recombinant GluSE (rec GluSE), purified GluV8 (native GluV8) of Staphylococcus aureus, and thermolysin-cleaved recombinant GluV8 (rec GluV8) on SDS-PAGE Separated. M represents a molecular weight marker. (B) and (C) Purified GluSE of Staphylococcus epidermidis (native GluSE), Thermolysin-treated recombinant GluSE (rec GluSE), Staphylococcus aureus purified GluV8 (native GluV8), Thermolysin-cleaved recombinant GluV8 (rec1 and rec2 GluV8) Protease activity was measured using fluorescent peptide (B) or azocasein (C) as a substrate. In advance, 10 μg / 50 μl of recombinant GluV8 was cleaved with 0.3 μg (rec1) or 1 μg (rec2) of thermolysin and used for activity measurement. Bars show standard deviations (n = 4-5). (B) used 0.25 μg and (C) used 1 μg of protein for activity measurement. In panel (B), the white column values are on the left scale, and the black columns are on the right scale. FIG. 5 shows the protease activity of the N-terminal deleted GluSE prosequence-GluV8 chimeric molecule. GluSE-GluV8 (Full) or a molecule starting from Ile49, Ile56, Asn61, Tyr64 (12 μg / 120 μl) with its N-terminal deleted stepwise is untreated (-), thermolysin (1 μg) treated (+ ) Later, (A) 0.5 μg of each sample was subjected to SDS-PAGE. Th indicates a control in which a solution containing only thermolysin (1 μg) was similarly treated. (B) Panel A shows protease activity of thermolysin untreated and treated samples (0.25 μg) measured with a fluorescent peptide substrate. Bars indicate standard deviation (n = 4). (C) Dose dependency of protease activity of a recombinant preparation untreated with thermolysin. FIG. 6 shows a comparison of the amino acid sequences of GluSE, GluV8 and GluSW. (A) The amino acid homology of the pre-sequence, pro-sequence and mature sequence of S. epidermidis GluSE, S. aureus GluV8, and S. warneri GluSW was compared. The GluV8 C-terminal repetitive sequence (amino acids 285-336) and the GluSW C-terminal Asn / Asp rich region (amino acids 283-316) were excluded from the comparison. (B) shows prepropeptide sequences of GluSE, GluV8, GluSW, and GluV8mut5. Lower case letters are sequences derived from the expression vector pQE60. Underlined amino acids indicate amino acids that may be self-digested. Two-residue deletions introduced to maximize homology are indicated by hyphens. The five amino acids (Asp36His, Glu62Gln, Glu65Ser, Ala67Pro, Asn68Ser) converted from GluV8 to GluSE in GluV8mut5 were marked with an asterisk. Arrows indicate processing sites (prepro boundaries and pro-mature region boundaries). FIG. 7 shows the expression of recombinant protease. E. coli lysates (5 μl) expressing recombinants were separated by SDS-PAGE. (A) CBB staining (b) Immunoblotting using His _6- tag specific monoclonal antibody (c) Talon affinity gel (20 μl) from lysate (1 ml) prepared from culture medium expressing recombinant protein (12 ml) 5 μl of the recombinant protein (40 μl) purified by 1 was separated by SDS-PAGE. The proteins in lanes 1-12 correspond to Nos. 1-12 in Table 2. Lane M: molecular weight marker. FIG. 8 shows the result of expressing a large amount of recombinant protease. (A) GluV8 (wild type, white circle) and GluV8mut5 ΔC (black circle), (b) GluSW (wild type, white circle) and GluV8mut5-SW (black circle) are expressed in 800 ml of culture medium, and lysate (60 ml) It was. Purified with a Talon affinity column (1.5 x 5 cm). The eluate was fractionated by 1 ml, and the absorbance at 280 nm was measured. The elution peak and its front and back (5 μl) were separated by SDS-PAGE (insert figure). FIG. 9 shows the activation of recombinant protease by thermolysin. 3.2 nmol of purified GluSE, GluV8mut5, GluV8mut5 ΔC and GluV8mut5-SW were not treated (lanes 1, 4, 7, 10, or 37 ° C, thermolysin 0.1 μg (lanes 2, 5, 8, 11) or 0.3 μg (lane 3) , 6,9,12) Incubate for 4 hours in the presence of recombinant protein / thermolysin molar ratios of 100/1 and 33/1, respectively, 3.2 pmol after heat denaturation in the presence of SDS (equivalent to 1 μg for GluSE) (B and c) The glutamate-specific protease activity of thermolysin-untreated or treated samples (0.8 pmol, 0.25 μg for GluSE) was measured, and c is column 4 in Fig. 3b. The same data was enlarged and plotted with the exception of -9. (D) Degradation activity was measured using azocasein as a substrate, and bd treated similarly with 0.1 μg (C1) and 0.3 μg (C2) thermolysin in the absence of recombinant. And a part of the control for measuring the activity. The sample in column 1-12 of d is identical to lane 1-12 of a.

SEQ ID NO: 1: GluV8 full-length sequence SEQ ID NO: 2: GluSE full-length sequence SEQ ID NO: 3: GluV8 pre-sequence SEQ ID NO: 4: GluV8 pro-sequence SEQ ID NO: 5: GluV8 pre-pro sequence SEQ ID NO: 6: GluSE pre-sequence SEQ ID NO: 7: GluSE pro-sequence No. 8: GluSE prepro sequence SEQ ID No. 9: primer (GluSE 5′-primer)
SEQ ID NO: 10: primer (GluSE 3′-primer)
Sequence number 11: Primer (GluV8 5'-primer)
SEQ ID NO: 12: primer (GluV8 3′-primer)
SEQ ID NO: 13: Primer (3′-primer for GluSE prepro region amplification)
SEQ ID NO: 14: Primer (5′-primer for GluV8 mature region amplification)
SEQ ID NO: 15: His tag SEQ ID NO: 16: Short mutant pro sequence SEQ ID NO: 17: GluV8 mutant prepro sequence SEQ ID NO: 18: GluSW full-length sequence SEQ ID NO: 19: Primer (GluSW 5′-primer)
SEQ ID NO: 20: primer (GluSW 3′-primer)
SEQ ID NO: 21: GluSW N-terminal sequence SEQ ID NO: 22: GluSW determined N-terminal sequence SEQ ID NO: 23: GluSW N-terminal sequence SEQ ID NO: 24: GluSW determined N-terminal sequence SEQ ID NO: 25: GluV8mut5-SW N-terminal SEQ ID NO: 26: determined N-terminal sequence of GluV8mut5-SW

Claims (10)

A promoter operable in Escherichia coli, and a polynucleotide in which a nucleotide encoding a prepro sequence of a glutamic acid-specific protease derived from Staphylococcus epidermidis and a nucleotide encoding S. aureus V8 protease , which is a mature protease , are linked in frame. A vector for expressing an inactive soluble protease in E. coli containing an expression cassette.   The expression vector according to claim 1, wherein the prepro sequence is a peptide having the amino acid sequence of SEQ ID NO: 8, or a peptide comprising an amino acid sequence consisting of at least the tyrosine at position 64 to the serine at position 66 of SEQ ID NO: 8. A promoter capable of operating in Escherichia coli, and a nucleotide encoding a mutant prepro sequence consisting of the amino acid sequence of SEQ ID NO: 17 in which 5 amino acid substitutions of the S. aureus V8 protease prepro sequence and a nucleotide encoding a mature protease are in frame. A vector that expresses an inactive soluble protease in E. coli, comprising an expression cassette containing a ligated polynucleotide , wherein the mature protease is Staphylococcus warneri glutamate-specific protease (EC 3.4.21.9). ), Factor Xa (EC 3.4.21.6), plasmin (EC 3.4.21.7), tissue-type plasminogen activator (EC 3.4.21.68), urokinase (EC 3.4.21.73), thrombin (EC 3.4.21.5), collagenase ( EC 3.4.24.3), Arginyl Gindi from periodontopathic bacteria Porphyromonas gingivalis Selected from the group consisting of In and lysyl Jinji pine, expression vectors. The expression vector according to any one of claims 1 to 3 , wherein the expression cassette further has a tag-encoding nucleotide linked in frame so that a tag is added to the C-terminus of the mature protease. The expression vector according to any one of claims 1 to 4 , wherein the promoter is a T5 promoter / lactose operator. The expression vector according to claim 1 or 3 , wherein the S. aureus V8 protease is deficient in a 12 amino acid repeat sequence of 3 amino acids consisting of C-terminal Pro-Asp-Asn or Pro-Asn-Asn. A transformant comprising the expression vector according to any one of claims 1 to 6 . The following process:
A step of culturing the transformant according to claim 7 ;
A method for producing an inactive protease, comprising: recovering a culture obtained in the culture step; and purifying a protease from the recovered culture. The following process:
A step of culturing the transformant according to claim 7 ;
Collecting the culture obtained in the culturing step;
A method for producing an active protease, comprising a step of purifying a protease from a collected culture, and a step of cleaving the prepro sequence by enzymatic treatment of the purified protease. A vector for producing an insoluble soluble protein in Escherichia coli containing a nucleotide encoding a mutated prepro sequence consisting of the amino acid sequence of SEQ ID NO: 17, which is obtained by substituting the prepro sequence of S. aureus V8 protease by 5 amino acids.