JP4252299B2 - Novel disulfide oxidoreductase and protein activation method using the enzyme - Google Patents

Description

【図面の簡単な説明】
【図１】本発明の新規タンパク質ＣａｔＡの発現ベクターｐＮＣｃａｔＡの構築方法及び構造を示す図。
【図２】発現ベクターｐＮＣｃａｔＡに組み込まれた本発明の新規タンパク質ＣａｔＡの発現を示すＳＤＳ−ＰＡＧＥの図面代用写真。
【図３】本発明の新規タンパク質ＣａｔＡのインスリンを用いた活性測定試験の結果を示す図。
【図４】本発明の新規タンパク質ＣａｔＡを用いた活性型ｈＥＧＦの生成を示すＳＤＳ−ＰＡＧＥの図面代用写真。
【図５】本発明の新規タンパク質ＣａｔＡをコードするＤＮＡとｈＥＧＦをコードするＤＮＡの共発現を行った培養上清をＳＤＳ−ＰＡＧＥに供し、ＣＢＢ染色を行った結果を示す図面代用写真。
【図６】本発明の新規タンパク質ＣａｔＡをコードするＤＮＡとｈＥＧＦをコードするＤＮＡの共発現を行った培養上清をＳＤＳ−ＰＡＧＥに供し、抗ＥＧＦ抗体を用いたウェスタンブロットを行った結果を示す図面代用写真。
【図７】遺伝子ｃａｔＡの塩基配列から推定される発現タンパク質のアミノ酸配列（配列番号１）を示す。
【図８】本発明に係る遺伝子ｃａｔＡの塩基配列（配列番号２）を示す。
【図９】本発明に係るジスルフィド酸化還元活性を有する新規タンパク質ＣａｔＡのアミノ酸配列（配列番号３）を示す。
【図１０】遺伝子ｃａｔＡに対応するタンパク質の２７位以降のアミノ酸配列（すなわち、新規タンパク質ＣａｔＡのアミノ酸配列）をコードするＤＮＡの塩基配列（配列番号４）を示す。
【図１１】プライマーＢｂＶＭ２の塩基配列（配列番号５）を示す。
【図１２】プライマーＢｂＶＭ２の塩基配列（配列番号６）を示す。
【図１３】プライマーＢｂ−ＯＸＲＥ２−５’の塩基配列（配列番号７）を示す。
【図１４】プライマーＢｂ−ＯＸＲＥ２−３’の塩基配列（配列番号８）を示す。
【図１５】プライマーＰｒｉｍｅｒ １（ｆｏｒｗａｒｄ）の塩基配列（配列番号９）を示す。
【図１６】プライマーＰｒｉｍｅｒ ２（ｂａｃｋｗａｒｄ）の塩基配列（配列番号１０）を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel disulfide oxidoreductase, a gene encoding the same, and a method for producing an active protein using the enzyme.
[0002]
[Prior art]
The genetic recombination technology has made it possible to produce a large amount of a biologically derived peptide or protein that has been extremely difficult to isolate as a trace component using microorganisms or animal cells. However, proteins produced using genetic recombination techniques often do not exhibit the original physiological activity of natural proteins, which is a major problem.
[0003]
One of the major causes of this is thought to be that the protein produced using genetic recombination technology has the same correct structure (sequence) as the protein with natural physiological activity in the primary structure (amino acid sequence). Although it has, the three-dimensional structure is different from the original three-dimensional structure having physiological activity.
[0004]
A disulfide bond formed between cysteine residues within a protein molecule and / or between molecules plays a very important role in the formation and maintenance of the three-dimensional structure of the protein and the accompanying expression of physiological activity. Proteins produced using genetic recombination techniques are deficient in this disulfide bond, that is, the disulfide bond that should be originally formed is not formed, or intramolecular and / or intermolecular cysteine residues In many cases, a disulfide bond at a position different from the original is formed. For this reason, it is known that the three-dimensional structure of a protein produced by genetic recombination differs from the structure of the original protein having physiological activity, or in some cases, insoluble aggregates of the protein are generated. Yes. This is thought to cause a decrease or disappearance of the physiological activity that the protein originally has.
[0005]
In addition, chemically synthesized polypeptides and proteins produced using cell extracts (Cell free system) also have the original correct three-dimensional structure, such as a relatively unfolded structure lacking disulfide bonds. It is often known that it often does not have, and therefore does not exhibit physiological activity.
[0006]
Disulfide bond formation / cleavage is a simple redox reaction of two cysteine residues. Thus, it can occur non-enzymatically in either direction under appropriate redox conditions. Therefore, a method is known in which a protein having a defect in a disulfide bond is converted into a protein having a correct disulfide bond by a chemical redox reaction. However, such a chemical oxidation-reduction reaction method cannot form an accurate disulfide bond with practical efficiency for a protein produced by gene recombination technology, particularly a protein having a large number of disulfide bonds. Impossible.
[0007]
Therefore, in order to obtain a protein having physiological activity by correcting the disulfide bond of a protein that does not exhibit physiological activity, a method widely attempted by those skilled in the art is known as a protein having disulfide redox activity (hereinafter referred to as “disulfide oxidation”). This may be referred to as “reductase”. For example, a method using thioredoxin as a disulfide oxidoreductase (for example, see Non-Patent Document 1), a method using protein disulfide isomerase (PDI) (for example, see Patent Document 1), and the like are known. Also known is a method for producing a protein having a correct disulfide bond by introducing a DNA encoding a disulfide oxidoreductase into a host organism and simultaneously expressing the desired recombinant protein and the disulfide oxidoreductase. (For example, refer to Patent Document 2).
[0008]
Major ones known as disulfide oxidoreductases include thioredoxin, human PDI (see, for example, Non-patent Document 2 and Patent Document 3), and Dsb derived from E. coli (see, for example, Non-patent Document 3 and 4) ) And other Dsb families. Furthermore, Bdb (for example, refer nonpatent literature 5) etc. are known as a thing derived from Bacillus subtilis.
[0009]
These disulfide oxidoreductases are known to have an amino acid sequence called a thioredoxin motif (CXXC) (C is a cysteine residue, X is an arbitrary amino acid residue) as a common feature. . This thioredoxin motif is regarded as an active site in a disulfide redox reaction (see, for example, Non-Patent Document 6). Furthermore, it is known that a known disulfide oxidoreductase is synthesized in vivo as a protein having a secretory signal sequence on the N-terminal side.
[0010]
[Non-Patent Document 1]
Pigiet VP and Schuster BJ, Proc. Natl. Acad. Sci. USA, 83, 7643-7647, (1986)
[0011]
[Patent Document 1]
JP 63-29496 A
[0012]
[Patent Document 2]
Japanese Patent Application Laid-Open No. 11-75879
[0013]
[Non-Patent Document 2]
Rapilajaniemi et al., EMBO J. 6, 643-649, (1987)
[0014]
[Patent Document 3]
Japanese Patent No. 2803309
[0015]
[Non-Patent Document 3]
EMBO J., 11, 55-62, (1992)
[0016]
[Non-Patent Document 4]
Cell, 67, 581-589, (1991)
[0017]
[Non-Patent Document 5]
Bolhuis et al. , J .; Biol. Chem. , 274, 24531-24538 (1999)
[0018]
[Non-Patent Document 6]
Muneo Sone, Ishiaki Ito, Protein, Nucleic Acid, Enzyme, 43, 1-10, (1998)
[0019]
[Problems to be solved by the invention]
As described above, disulfide oxidoreductases such as thioredoxin and PDI are used for activating proteins that do not exhibit physiological activity. However, depending on the type of protein that does not exhibit physiological activity, it could often not be activated using any of the known disulfide oxidoreductases. One reason for this is that each disulfide oxidoreductase has substrate specificity, and the disulfide oxidoreductase has different proteins that can act.
[0020]
Therefore, in order to enable activation using various disulfide oxidoreductases even for various proteins that do not exhibit physiological activity, provision of further novel disulfide oxidoreductases has been demanded.
[0021]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have focused on Brevibacillus choshinesis, which is effectively used as a host in the production of recombinant proteins, and its genome. From which a novel protein gene presumed to have disulfide redox activity was found and named catA. The base sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the protein deduced from the sequence is shown in SEQ ID NO: 1.
[0022]
Furthermore, the present inventors presume that it encodes the mature part excluding the secretory signal sequence of the protein corresponding to the gene in the base sequence (SEQ ID NO: 2) of the above-described novel gene catA according to the present invention. An expression vector into which DNA having the base sequence (SEQ ID NO: 4) was inserted was constructed, and Brevibacillus choshinensis was further transformed with the expression vector. Furthermore, by culturing the transformant, the DNA was expressed in a large amount to obtain the novel protein CatA (SEQ ID NO: 3) of the present invention.
[0023]
Furthermore, the present inventors measured disulfide redox activity using insulin for this new protein CatA, and as a result, it was confirmed that the new protein CatA of the present invention has disulfide redox activity.
[0024]
Furthermore, the present inventors obtained active hEGF by allowing this novel protein CatA to act on inactive human epidermal growth factor (hEGF), and the novel protein CatA of the present invention activates the inactive protein. To show that it is available.
[0025]
Furthermore, the present inventors co-expressed CatA-encoding DNA and hEGF-encoding DNA using Brevibacillus choshinensis as a host, and as a result, the production amount of active recombinant hEGF increases. As a result, it was demonstrated that the protein CatA of the present invention can be used to increase the production amount of a recombinant protein having physiological activity by co-expression with other recombinant proteins. Thus, the present inventors have completed the present invention.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The amino acid sequence of the novel protein CatA having disulfide redox activity of the present invention is exemplified in SEQ ID NO: 3. Nothing having 40% or more homology with this amino acid sequence has been reported, and therefore the protein CatA of the present invention is a novel protein.
[0027]
Furthermore, even if the protein is composed of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 3 is substituted, deleted, inserted or added, as long as it has disulfide redox activity, all of the disulfides of the present invention It is included in the protein CatA having redox activity. Here, “the amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added” differs depending on the type of amino acid residue and the position of the amino acid residue in the three-dimensional structure of the protein. It means an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more homology with respect to the entire amino acid sequence of 3.
[0028]
A polypeptide comprising an amino acid sequence in which one or several amino acids in the above-mentioned specific amino acid sequence is substituted, deleted, inserted or added is, for example, site-directed mutagenesis method, gene homologous recombination method, primer extension method , And a method well known to those skilled in the art, such as PCR, can be easily prepared.
In this case, in order to have substantially the same function, among the amino acids constituting the polypeptide, homologous amino acids (polar / nonpolar amino acids, hydrophobic / hydrophilic amino acids, positive / negative charged amino acids, Substitution between aromatic amino acids and the like is possible. In order to maintain a substantially equivalent function, it is desirable to retain amino acids in the functional domain included in each polypeptide of the present invention.
[0029]
In addition, SEQ ID NO: 4 illustrates the base sequence of DNA encoding the protein CatA of the present invention. Nothing having 40% or more homology with this base sequence has been reported, and therefore the DNA encoding the protein of the present invention is a novel DNA. Furthermore, even if the DNA encoding a protein comprising an amino acid sequence in which one or several amino acids in the amino acid sequence shown in SEQ ID NO: 3 is substituted, deleted, inserted or added, the encoded protein is disulfide oxidized. All are included in the DNA encoding the protein of the present invention as long as they have reducing activity. Here, the meaning of “amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added” is the same as in the case of the protein of the present invention described above.
[0030]
Further, as described above, the DNA of the gene encoding the protein CatA of the present invention indicates a DNA having the base sequence shown in SEQ ID NO: 4, or a probe prepared from the entire base sequence of this DNA or a part thereof. And a DNA encoding a protein that hybridizes under stringent conditions and has disulfide redox activity.
[0031]
In the present invention, “under stringent conditions” means that the degree of homology between base sequences is, for example, an average of about 80% or more, preferably about 90% or more, more preferably about 95% on the whole. It means the condition that a hybrid is specifically formed only between base sequences having high homology, such as% or more. Specifically, for example, the conditions include a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and pH of 6 to 8 at a temperature of 60 to 68 ° C.
[0032]
Hybridization may be performed by a method known in the art, such as, for example, the method described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al, (1987)). In addition, when a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
[0033]
Furthermore, the novel protein CatA of the present invention can be obtained by appropriately selecting standard genetic recombination techniques known to those skilled in the art and using them in combination. For example, it can be performed using the method described in Molecular Cloning: Laboratory Manual 2nd Edition (Sambrook et al, Cold Spring Harbor Laboratory Press, New York (1989)).
[0034]
The DNA encoding the protein CatA of the present invention is, for example, a Brevibacillus choshinensis genomic DNA library prepared as shown in Example 1 to be described later. From this library, for example, represented by SEQ ID NO: 4 It can be prepared by a hybridization method using a DNA fragment having a part of the base sequence as a probe or a PCR method using the DNA fragment as a primer. Alternatively, based on the base sequence, DNA encoding the protein CatA of the present invention can be obtained by a nucleic acid chemical synthesis method known to those skilled in the art, but is not limited thereto. The sequences shown in SEQ ID NOs: 1, 2, 3, and 4 are also shown in FIGS.
[0035]
The host used for the expression of the DNA encoding CatA is not particularly limited as long as it can express the DNA encoding CatA. As the host, any of bacteria, yeast, mold, animal cells and the like can be used, preferably bacteria, and more preferably Brevibacillus bacteria. Particularly preferred hosts include Brevibacillus choshinensis HPD31 (FERM BP-6863) and its mutant Brevibacillus choshinensis HPD31-S5 (FERM BP-6623).
[0036]
The vector into which the DNA encoding CatA is inserted is not particularly limited as long as it can be replicated in the host. However, when a Brevibacillus bacterium is used as a host, pNY301 (Japanese Patent Laid-Open No. 10-295378) is particularly preferable. ) And pNCMO2 (Yashiro et al, Protein Expression and Purification 23, 113-120 (2001)).
[0037]
In order to insert DNA encoding CatA into a vector, for example, purified DNA containing a base sequence encoding CatA is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or a multiple cloning site of an appropriate vector. However, it is not limited to this. Further, since it is necessary to be incorporated into a vector so that the function of the DNA encoding CatA is exhibited, the vector may include a promoter, DNA encoding CatA, a terminator, a ribosome binding sequence, and the like. Good.
[0038]
The method of introducing the expression vector into the host is also not particularly limited, but when a Brevibacillus bacterium is used as the host, electroporation (Takagi et al, Agric. Biol. Chem., 53 , 3099-3100 (1989)).
[0039]
The culture method of the host used for the expression of the DNA encoding CatA is not particularly limited as long as it can express the DNA encoding CatA, but is particularly preferable when a Brevibacillus bacterium is used as the host. Examples of conditions include the TMN medium used in the following examples, 30 ° C., and 2 to 4 days.
[0040]
Furthermore, when the expressed CatA is isolated and purified, it can be carried out by using methods known to those skilled in the art, singly or in combination. For example, it is possible to use ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, or the like as appropriate, alone or in combination.
[0041]
In the method for activating a protein that does not exhibit physiological activity by acting the protein CatA having disulfide redox activity according to the present invention, the protein that does not exhibit physiological activity to which the method can be applied is produced by a gene recombination technique. Various proteins, various polypeptides chemically synthesized by a peptide synthesizer, various proteins synthesized by an in vitro protein synthesis system, and their contents. . In addition to the protein itself, the protein produced by genetic recombination technology is the biologically inactive material that is separated from and separated from the same production culture (transformant culture). Examples include proteins and their contents.
[0042]
Furthermore, in the protein activation method of the present invention, the amount of CatA that acts on the protein is not particularly limited. CatA can be acted on a protein by adding a solution in which CatA is dissolved in a buffer solution to the protein or solubilizing the protein with an appropriate denaturing agent and then adding CatA to the protein solution. Good. The reaction temperature is preferably 0 to 40 ° C., and the reaction time is suitably 1 to 5 hours.
[0043]
In addition, CatA of the present invention can be directly caused to act on a protein that does not exhibit physiological activity. However, when acting on an insoluble protein, the protein is first dissolved in a reducing denaturation solution, and further in the presence of a disulfide compound. CatA is preferably allowed to act. The reduced denaturing solution is preferably 0.1 to 0.2 M dithiothreitol, 1 to 6 M guanidine hydrochloride solution, or 1 to 8 M urea solution. The concentration of the protein that does not exhibit physiological activity dissolved in the denaturing solution is 0.00. About 1 to 40 mg / ml is appropriate. The disulfide compound is preferably oxidized glutathione or oxidized dithiothreitol. The disulfide compound must be added at a concentration exceeding the molar concentration of the sulfhydryl compound in the entire reaction solution.
[0044]
Furthermore, when CatA is reacted with a protein that does not exhibit physiological activity, CatA may be contacted by passing it through a protein solution immobilized in a closed space with an MF membrane or the like and dissolved in a buffer solution or the like. Is immobilized by adsorbing CatA on a carrier such as HP-20 (manufactured by Mitsubishi Chemical Corporation), which is a porous resin, to form an immobilized column, and a protein solution is supplied to this immobilized column to contact CatA You may let them.
[0045]
In addition, the protein CatA of the present invention can be used to increase the production amount of a recombinant protein having physiological activity by co-expressing a DNA encoding CatA and a DNA encoding another protein. is there. In this case, a preferred method for co-expressing DNA encoding CatA and DNA encoding another protein includes DNA encoding CatA and DNA encoding another protein, as shown in the Examples. This is a method of co-expression using a host transformed simultaneously with another expression vector.
[0046]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
[0047]
(Example 1: Cloning of a novel gene catA)
Brevibacillus choshinensis HPD31-S5 (FERM BP-6623) was cultured under conditions of 5 ml TMN, 30 ° C., 4 days. TMN medium is a medium in which neomycin is added to TM medium at a concentration of 50 μl / ml. The composition of TM medium is 1% polypeptone, 0.5% meat extract, 0.2% yeast extract, 1% glucose, FeSO _Four ・ 7H ₂ O 0.001%, MnSO _Four ・ H ₂ O 0.001%, ZnSO _Four ・ 7H ₂ O 0.001%, pH 7.0. After completion of the culture, the culture solution was centrifuged (8000 rpm, 30 minutes) at room temperature to collect bacteria. Next, the collected bacterial cells were washed twice with a 10 mM Tris (pH 7.5) -1 mM EDTA solution and then separated into a phenol layer to prepare a chromosomal DNA of Brevibacillus choshinensis HPD31-S5.
[0048]
Furthermore, primers were designed with reference to the nucleotide sequences of both the yneN of Bacillus subtilis and BH1522 genes of Bacillus halodurans presumed to encode a protein having disulfide redox activity, and Brevibacillus choshinensis HPD31-S5 obtained above was used. PCR was performed using the chromosomal DNA as a template to obtain an amplification product of about 180 bp. The following primers were used.
[0049]
BbVM2 (forward primer): 5′-CATKTTTGGGRCKWCDTGTG-3 ′ (SEQ ID NO: 5: FIG. 11)
BbVM4 (backward primer): 5′-GTCRAKVAVAAYSGGDAASGT-3 ′ (SEQ ID NO: 6: FIG. 12)
(In the above sequences, R is A or G, Y is C or T, W is A or T, S is G or C, K is G or T, D is A or G or T, V Represents A, C, or G, respectively, and the forward primer BbVM2 corresponds to the base sequence from the 192nd to the 212th base of the Bacillus subtilis yneN gene ORF, and the backward primer BbVM4 also corresponds to the 355th to 375th sequence. Corresponding.)
[0050]
PCR was performed at 1.95 ° C. for 9 minutes, 2.94 ° C. for 30 seconds, 3.40 ° C. for 60 seconds, 4.72 ° C. for 90 seconds, 5.60 ° C. for 10 minutes: 2 to 4 were performed under 40 cycles. It was.
[0051]
Furthermore, when this PCR product was cloned into EcoRV digested pBR322 and the nucleotide sequence was decoded, it was confirmed that the PCR product was a partial sequence of the target gene from the similarity to both genes yneN and BH1522. Next, Southern analysis of the PstI-digested brevis chromosome was performed using the amplification product as a probe, and the result indicating that the target gene was contained in an approximately 2.5 kbp fragment was obtained. Therefore, the following two primers were designed from the internal sequence of the amplified product.
[0052]
Bb-OXRE2-5 ′: 5′-CAATGTGACCAATAGTGATAGTCCC-3 ′ (SEQ ID NO: 7: FIG. 13)
Bb-OXRE2-3 ′: 5′-CCTGATCTTTGTTACTCTCCGTAC-3 ′ (SEQ ID NO: 8: FIG. 14)
[0053]
Chromosomal DNA of Brevibacillus choshinensis HPD31-S5 digested with the restriction enzyme PstI was subjected to agarose electrophoresis, a band corresponding to a size of 2.6 kbp was cut out from the gel, and self-ligation was performed. Using this self-ligated DNA as a template, PCR was carried out using the above two primers to obtain a 2.6 kbp PCR product. Furthermore, the base sequence of this product was decoded, and it was confirmed that the full length of the target gene was included. The novel gene presumed to encode a protein having this 531 bp disulfide redox activity was named catA. The base sequence of the gene catA is shown in SEQ ID NO: 2 (FIG. 8). The amino acid sequence of the protein corresponding to catA deduced from the base sequence of gene catA is shown in SEQ ID NO: 1 (FIG. 7). In this 177 amino acid protein, the thioredoxin motif is present at positions 73-76.
[0054]
(Example 2: Production of CatA by gene recombination method)
Further, in the protein corresponding to this novel gene catA, it is presumed that the position 1 to 26 is a secretory signal sequence and the 27th and subsequent alanine is a mature body, and it is presumed to be a mature body as shown below. Attempted production by genetic recombination for the part. However, whether the protein is actually a secreted protein in Brevibacillus choshinensis, or if it is secreted, the secret signal cleavage site must be before alanine.
[0055]
Based on the base sequence of the novel gene catA obtained in Example 1, the following two primers are prepared, PCR is performed using the chromosomal DNA of Brevibacillus choshinensis HPD31-S5 as a template, and the mature form of the protein corresponding to the gene catA A gene encoding the alanine after the 27th position was amplified. PCR was performed by repeating a cycle of 95 ° C for 1 minute, 54 ° C for 1 minute, and 72 ° C for 1 minute 26 times.
[0056]
Primer 1 (forward): 5′-GGTCCGCTGCAGTCCGTAGCAGAGGTC-3 ′ (SEQ ID NO: 9: FIG. 15)
Primer 2 (backward): 5′-CGACGGATCCTATGGTTTTTCATTCAAAAGCTTGC-3 ′ (SEQ ID NO: 10: FIG. 16) In addition, Primer 1 has a PstI site (CTGCAG) and Primer 2 has a BamI site (BamH site). GGATCC).
[0057]
Next, the PCR product obtained above was digested with PstI and BamHI, then ligated to the expression vector pNCMO2 (Yashiro et al, Protein Expression and Purification 23, 113-120 (2001)) treated with the same enzyme, and the expression plasmid pNCcatA Got. The construction method and structure of the expression vector pNCcatA are shown in FIG.
[0058]
In this pNCcatA, DNA encoding alanine after position 27 of the amino acid sequence of the protein corresponding to the gene catA leads to the secretory signal sequence of the brevis cell wall protein contained in pNCMO2. Therefore, CatA secreted from HPD31-S5 is expected to be alanine at the N-terminus of the secreted protein because the secretory signal sequence is cleaved before alanine.
[0059]
Furthermore, this expression plasmid pNCcatA was introduced into Brevibacillus choshinensis HPD31-S5 (FERM BP-6623) by electroporation (Takagi et al, Agric. Biol. Chem., 53, 3099-3100 (1989)). The obtained transformant was named Brevibacillus choshinensis HPD31-S5 / pNCcatA, and deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center under the accession number FERM P-19124.
[0060]
Then, this transformant Brevibacillus choshinensis HPD31-S5 / pNCcatA (FERM P-19124) was cultured at 30 ° C. in 3 ml of TMN medium for 2 days. After completion of the culture, the culture solution was centrifuged at room temperature (8000 rpm, 30 minutes), and 4 μl of the supernatant was subjected to SDS-PAGE. The result of SDS-PAGE is shown in FIG. As shown in the result of SDS-PAGE, a staining band is present at a position corresponding to a molecular weight of about 17 kDa of the amino acid residue after the 27th position of the protein corresponding to the gene catA, and 27 of the protein corresponding to the gene catA. Of the present invention, which is a protein having the expression of DNA encoding the amino acid sequence after position (the base sequence is shown in SEQ ID NO: 4 (FIG. 10)) and the amino acid sequence after position 27 of the protein corresponding to the gene catA. Secretory production of the novel protein CatA was confirmed. Further, N-terminal analysis of CatA produced by secretion was performed to confirm that the N-terminal was alanine. The amino acid sequence of CatA obtained as described above is shown in SEQ ID NO: 3 (FIG. 9). In FIG. 2 (photograph substituted for a drawing showing the SDS-PAGE pattern), 1 is the result of the culture supernatant of the host fungus, and 2 is the result of the culture supernatant of the transformant transformed with the host fungus. is there.
[0061]
(Example 3: Activity measurement of CatA)
Furthermore, according to the following procedure, using the novel protein CatA of the present invention obtained in Example 2 above, disulfide bond formation / cleavage reaction by oxidation-reduction of the cysteine residue of insulin was performed, and the disulfide of the novel protein CatA of the present invention The redox activity was evaluated.
[0062]
The CatA enzyme preparation was prepared by the following procedure. The transformant Brevibacillus choshinensis HPD31-S5 / pNCcatA (FERM P-19124) prepared in Example 2 was cultured under conditions of 5 ml TMN medium, 30 ° C., 4 days. After completion of the culture, the culture broth was centrifuged at room temperature (8000 rpm, 30 minutes), and 100 μl of the supernatant was used as a CatA enzyme preparation.
[0063]
A substrate solution in which insulin that causes an enzyme reaction was dissolved was prepared as follows. First, 25 mg of insulin was dissolved in 2 ml of 50 mM Tris-HCl, pH 8.0, then 1N HCl was added to adjust the pH to 2-3, and then 1N NaOH was immediately added to return the pH to the original 8.0. Thereafter, water was added to make a total volume of 2.5 ml to obtain a 10-fold concentrated stock solution. When measuring the activity, this stock solution was diluted 10-fold with 0.1 M phosphate buffer (pH 7.0) containing 2 mM EDTA.
[0064]
500 μl of the substrate solution in which the insulin prepared above was dissolved was placed in a glass cuvette for a spectrophotometer, and further 100 μl of the CatA enzyme preparation obtained above and 5 μl of 0.1 M DTT were added and reacted at 25 ° C. The course of the reaction is O.D. D. Followed by observation of the turbidity change of the reaction at 650. As a control, a pNCMO2 solution was used. The measurement results are shown in FIG.
[0065]
As shown in the measurement results of FIG. 3, an increase in the turbidity of the reaction solution was observed with the addition of CatA. This is presumably because addition of CatA formed a disulfide bond within the insulin molecule or between the insulin molecules, resulting in aggregation between the insulin molecules.
This result shows that the novel protein CatA of the present invention has disulfide redox activity.
[0066]
(Example 4: Conversion of hEGF having no physiological activity using CatA to hEGF having physiological activity)
(1) Preparation of inactive recombinant human epidermal growth factor (hEGF)
Brevibacillus choshinensis carrying the hEGF expression plasmid highly produces human epidermal growth factor (hEGF), but a part of the produced hEGF may be multimerized by forming a disulfide bond between hEGF molecules. Reported by Miyauchi et al. (Miyauchi et al, Journal of Industrial Microbiology and Biotechnology, 21, 208-214, (1998)). This multimeric hEGF is known to be an inactive hEGF that does not have the physiological activity that hEGF originally has, and a method for separating multimeric hEGF from a culture solution is also reported in the same literature. . Therefore, multimeric hEGF, which is an inactive hEGF, was prepared by the following method based on the description in the document.
[0067]
Brevibacillus choshinensis HPD31-S5 carrying the human epidermal growth factor (hEGF) expression plasmid pHT110 EGF described in the above document was subjected to liquid culture for 65 hours under the culture conditions described in that document, and the culture supernatant was recovered by centrifugation. Subsequently, the supernatant was adjusted to pH 3.0 with 6M hydrochloric acid to specifically aggregate multimeric hEGF, and this precipitate was collected by centrifugation. The precipitate was dissolved in a buffer solution of 0.1M Tris-HCl (pH 7.0), adjusted to pH 3.0 again with 6M hydrochloric acid, and the precipitate was collected by centrifugation. Further, the precipitate was suspended in distilled water, adjusted to pH 7.0 with sodium hydroxide solution, and then lyophilized to prepare multimeric hEGF, which is an inactive hEGF.
[0068]
(2) Conversion of inactive hEGF to active hEGF
Using inactive hEGF prepared by the above method, activation of inactive protein by CatA was tested by the following procedure. First, erythromycin and chloramphenicol are added to an inactive hEGF solution (5 mg / ml, pH 6.0) to a concentration of 100 μg / ml and neomycin at a concentration of 50 μg / ml, respectively, and 100 mM MES buffer (pH 6. 0) was added to prepare a solution with a hEGF concentration of 2 mg / ml. This solution was used as a reaction substrate for the enzyme reaction.
[0069]
The sample solution to be evaluated was prepared as follows. Brevibacillus choshinensis HPD31 was used as the host strain. HPD31 carrying the CatA expression vector pNCcatA, HPD31 carrying pNCMO2 and HPD31 not carrying a plasmid, the above three types of HPD31 are HP + 31 carrying a plasmid in a TM + neomycin medium, and HPD31 without a plasmid contains no drug. The cells were cultured in TM medium for 2 days. After completion of the culture, each culture solution is diluted by adding an appropriate amount of a centrifugal supernatant of the same culture solution. D. Adjusted to 660 = 2. This diluted solution was used as a sample solution.
[0070]
The activity was measured by the following procedure using the prepared substrate solution and sample solution. 100 μl of the sample solution and 1 μl of 200 mM reduced glutathione were added to 99 μl of the substrate solution, and the reaction was started at 35 ° C. 1, 3, 6 and 12 hours after the start of the reaction, the reaction solution was centrifuged, 50 μl of the supernatant was mixed with 50 μl of SDS-PAGE sample buffer not containing mercaptoethanol, heated at 70 ° C. for 10 minutes, and then 20 μl. SDS-PAGE was performed using The result of SDS-PAGE is shown in FIG. In FIG. 4 (drawing substitute photograph showing an SDS-PAGE pattern), 1, 2, 3, and 4 on the horizontal axis indicate reaction solutions 1, 3, 6, and 12 hours after the start of the reaction, respectively.
[0071]
As shown in FIG. 4, as a result of SDS-PAGE, a band corresponding to the molecular weight of active hEGF was obtained in the reaction solution to which the sample solution containing CatA was added, and the band became deeper as the reaction time passed. . Therefore, it was demonstrated that the protein CatA of the present invention has a function of converting a protein having physiological activity by acting on a protein that does not exhibit physiological activity.
[0072]
(Example 5: Change in production amount of active hEGF by co-expression of hEGF and CatA) Furthermore, hEGF expression plasmid pHT110 EGF and CatA expression plasmid pNCcatA were introduced into the same host, and the effect of CatA coexpression on hEGF production was observed. investigated. Brevibacillus choshinensis HPD31-S5 was used as the host. The host was transformed by mixing the expression plasmids pHT110 EGF and pNCcatA at a ratio of 1: 1, and then introducing the expression plasmid into the host by electroporation. After transformation, the cells were applied to 2SLEN plate medium. 2SLEN medium is a medium in which erythromycin, a resistance marker for pHT110 EGF, is added to 2SL medium at a concentration of 10 μl / ml, and neomycin, a resistance marker for pNCcatA, is added at a concentration of 50 μl / ml. The composition of the 2SL medium is 4% polypeptone S, 0.5% yeast extract (Lalman), FeSO _Four ・ 7H ₂ O 0.001%, MnSO _Four ・ 4H ₂ O 0.001%, ZnSO _Four ・ 7H ₂ O 0.001%, pH 7.2. The 2SLEN medium coated with the cells was placed at 30 ° C. for 2 days, and the cells were cultured. The transformant constituting the appearing colony retained both pHT110 EGF and pNCcatA plasmids. After completion of the culture, the culture supernatant was subjected to SDS-PAGE under reducing and non-reducing conditions, and Western blotting using CBB staining and anti-hEGF antibody was performed. The results of CBB (Coomassie Brilliant Blue) staining on SDS-PAGE are shown in FIG. 5 (drawing substitute photo), and the results of Western blotting using an anti-hEGF antibody are shown in FIG. 6 (drawing substitute photo). Show.
[0073]
Furthermore, in order to know the amount of active hEGF in the supernatant, a quantitative analysis was separately performed by HPLC. As a result of HPLC analysis, the value of 0.65 g / l was obtained when the average of the control coexpressed CatA with respect to 0.41 g / l. That is, about 60% of promoting effect was recognized by co-expression.
[0074]
As a result of the above-described experiment, the novel protein CatA of the present invention can reduce the production amount of a recombinant protein having physiological activity by co-expressing a DNA encoding CatA and a DNA encoding another recombinant protein. It has been demonstrated to have an increasing function.
[0075]
As a result of the above experiment, the novel protein CatA of the present invention has a disulfide redox activity, and further has a physiological activity when a protein that does not exhibit the physiological activity is allowed to act on a protein that does not exhibit the physiological activity. Further, the novel protein CatA of the present invention has physiological activity by co-expressing DNA encoding CatA and DNA encoding another recombinant protein. It has been demonstrated that it has the function of increasing the amount of recombinant protein produced.
[0076]
【The invention's effect】
The present invention provides a novel protein CatA having disulfide redox activity derived from a bacterium belonging to the genus Brevibacillus, a DNA encoding the protein, and a method for activating a protein not exhibiting physiological activity using the enzyme. Since the protein CatA of the present invention has a disulfide redox activity, it can be used to activate a protein that does not exhibit physiological activity by acting on a protein that does not exhibit physiological activity. Furthermore, the protein CatA of the present invention is used for increasing the production amount of a recombinant protein having physiological activity by co-expressing a DNA encoding CatA and a DNA encoding another recombinant protein. Is possible.
[0077]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the construction method and structure of an expression vector pNCcatA for the novel protein CatA of the present invention.
FIG. 2 is a drawing-substituting photograph of SDS-PAGE showing the expression of the novel protein CatA of the present invention incorporated into the expression vector pNCcatA.
FIG. 3 is a view showing the results of an activity measurement test using insulin of the novel protein CatA of the present invention.
FIG. 4 is a drawing-substituting photograph of SDS-PAGE showing the production of active hEGF using the novel protein CatA of the present invention.
FIG. 5 is a drawing-substituting photograph showing the results of subjecting a culture supernatant co-expressed with a DNA encoding the novel protein CatA of the present invention and a DNA encoding hEGF to SDS-PAGE and performing CBB staining.
FIG. 6 shows the results of subjecting a culture supernatant in which DNA encoding the novel protein CatA of the present invention and DNA encoding hEGF to co-expression to SDS-PAGE and Western blotting using an anti-EGF antibody. Drawing substitute photo.
FIG. 7 shows the amino acid sequence (SEQ ID NO: 1) of the expressed protein deduced from the base sequence of gene catA.
FIG. 8 shows the base sequence (SEQ ID NO: 2) of the gene catA according to the present invention.
FIG. 9 shows the amino acid sequence (SEQ ID NO: 3) of a novel protein CatA having disulfide redox activity according to the present invention.
FIG. 10 shows the base sequence (SEQ ID NO: 4) of a DNA encoding the amino acid sequence after position 27 of the protein corresponding to the gene catA (that is, the amino acid sequence of the novel protein CatA).
FIG. 11 shows the base sequence of primer BbVM2 (SEQ ID NO: 5).
FIG. 12 shows the base sequence of primer BbVM2 (SEQ ID NO: 6).
FIG. 13 shows the base sequence (SEQ ID NO: 7) of primer Bb-OXRE2-5 ′.
FIG. 14 shows the base sequence (SEQ ID NO: 8) of primer Bb-OXRE2-3 ′.
FIG. 15 shows the base sequence (SEQ ID NO: 9) of primer Primer 1 (forward).
FIG. 16 shows the base sequence (SEQ ID NO: 10) of primer Primer 2 (backward).

Claims (8)

Protein CatA of the following (a) or (b).
(A) A protein comprising the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
(B) A protein having an amino acid sequence in which one or several amino acid residues are substituted, deleted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing and having disulfide redox activity. DNA encoding the following protein (a) or (b):
(A) A protein comprising the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
(B) A protein having an amino acid sequence in which one or several amino acid residues are substituted, deleted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing and having disulfide redox activity. The DNA of the gene according to claim 2, which is DNA shown in the following (A) or (B).
(A) DNA consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing.
(B) A DNA that hybridizes with a probe consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing under a stringent condition and encodes a protein having disulfide redox activity.   Brevibacillus choshinensis HPD31-S5 / pNCcatA (FERM P-19124), a transformant obtained by transformation with an expression vector containing a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 in the Sequence Listing.   The transformant obtained by transforming a host with the expression vector containing the DNA according to claim 2 or 3 is cultured, and the protein CatA according to claim 1 is recovered from the culture. A method for producing the protein CatA described in 1.   A method for activating human epidermal growth factor (hEGF), which comprises allowing the protein CatA according to claim 1 to act on human epidermal growth factor (hEGF) that does not exhibit physiological activity.   By transforming a host with an expression vector containing DNA encoding human epidermal growth factor (hEGF) and an expression vector containing the DNA according to claim 2 or 3, and culturing the host, A gene group having physiological activity, characterized by simultaneously expressing DNA encoding human epidermal growth factor (hEGF) and the DNA according to claim 2 or 3 and recovering human epidermal growth factor (hEGF) A method for producing recombinant human epidermal growth factor (hEGF).   The method according to claim 5 or 7, wherein the genetically modified host is Brevibacillus choshinensis.

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