JPH1175876A - Production of new microbial transglutaminase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a microbial transglutaminase, produced by a gene recombination technique and freed of a methionine residue. SOLUTION: This protein contains a sequence ranging from a serine residue at the 2-position to a proline residue at the 331-position in an amino acid sequence represented by the formula and has the transglutaminse activity. The amino acid sequence at the N-terminal thereof corresponds to the serine at the 2-position of the amino acid sequence represented by the formula. A codon used is changed for Escherichia coli and a multiple copy vector (pUC19) and a strong promoter (trp promoter) are preferably adopted to express a microbial transglutaminase in the form freed of an aspartic acid residue which is the N-terminal amino acid of the microbial transglutaminase. Thereby, the protein is obtained in a large amount.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a protein having transglutaminase activity, and a D-protein encoding the protein.
More particularly, the present invention relates to a method for producing a protein having transglutaminase activity by genetic engineering techniques.

[0002]

2. Description of the Related Art Transglutaminase is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group in a peptide chain of a protein. When this enzyme is allowed to act on a protein, an ε- (γ-Glu) -Lys cross-linking reaction and a displacement reaction of Gln into Glu by deamidation may occur. This transglutaminase is used for the production of gelled foods such as jelly, yogurt, cheese, gel cosmetics, etc., and the improvement of meat quality (Japanese Patent Publication No. 1-50382). Further, it is an enzyme having high industrial utility, such as being used for the production of heat-stable microcapsule materials and carriers for immobilized enzymes. As for transglutaminase, those derived from animals and those derived from microorganisms (microvial transglutaminase: hereinafter referred to as “MTG”) are known.

[0003] The former is a calcium ion-dependent enzyme, which is distributed in animal organs, skin, blood and the like. For example, guinea pig liver transglutaminase (K. Ikura
et al. Biochemistry 27 2898 (1988)), human epidermal keratinocyte transglutaminase (MA Phillips e
Natl. Acad. Sci. USA 87 9333 (1990)),
Human blood coagulation factor XIII (A. Ichinose et al. Biochemis
try 25 6900 (1990)). Regarding the latter, calcium-independent ones have been found from bacteria of the genus Streptoverticillium. For example, Streptoverticillium griseocarneum (Streptoverticillium
griseocarneum) IFO 12776, Streptoverticillium cinnamoneum
sub sp. cinnamoneum) IFO 12852, Streptoverticillium mobaraens
ense) IFO 13819 and the like (JP-A-64-2747).
1).

As a result of peptide mapping and gene structure analysis, it has been found that the primary structure of transglutaminase produced by these microorganisms has no homology to that derived from animals (European Patent Publication 0 481).
504 A1). Microbial transglutaminase (MT
G) has been problematic in terms of supply amount, efficiency, etc. since it is produced from a culture of the above fungi and the like through a purification operation. Production methods using genetic engineering techniques have also been attempted.
It is based on secretion expression in microorganisms such as Escherichia coli (E. coli) and yeast (Japanese Patent Laid-Open No. 5-199883).
After expressing MTG as an inactive fusion protein inclusion body in Escherichia coli, the inclusion body is solubilized with a protein denaturing agent, and then subjected to denaturing agent treatment to regenerate the active MTG.
(JP-A-6-30771).

[0005] These methods have a problem in actual industrial production. That is, in the case of secretory expression in microorganisms such as E. coli and yeast, the expression level is extremely small, and when expressed as a fusion protein inclusion body in E. coli, an expensive enzyme is required for cleavage. It is. On the other hand, when a heterologous protein is secreted using genetic engineering,
It is generally known that the expression level is small. On the other hand, it is known that when a heterologous protein is expressed in cells using E. coli, the amount of expression is large, but often expressed as an inactive protein inclusion body. This protein inclusion body requires an operation of solubilizing with a denaturing agent and regenerating the activity through a denaturing agent treatment.

Furthermore, in the case of expression in E. coli, it has been found that usually, the N-terminal methionine residue of a natural protein obtained as a result of translation from a gene is efficiently cleaved by methionine aminopeptidase. But,
In the case of a foreign protein, this N-terminal methionine residue is not always cleaved. Heretofore, as a method for obtaining a protein not containing an N-terminal methionine residue, a protein having a methionine residue at the N-terminus by cyanogen bromide or a fusion protein in which a peptide is added via a methionine residue has been prepared, A chemical method that specifically decomposes at the site of the methionine residue, or a fusion peptide in which a recognition sequence for a specific site-specific protease is inserted between an appropriate peptide and a target peptide, There is an enzymatic method of specifically hydrolyzing.

[0007] However, the former method cannot be applied to those containing a methionine residue in the protein sequence, and the target protein may be denatured during the reaction. Further, even in the latter case, if a sequence that is easily degraded is present in the protein sequence, it cannot be applied because the yield of the target protein is reduced, and further, the use of such a degrading enzyme is costly. However, this method is not suitable for actual industrial production of proteins.

[0008]

The conventional MTG manufacturing method has many points to be improved in terms of supply amount, supply cost, and the like. That is, in the case of secretory expression in E. coli, yeast and the like, there is a problem that the expression level is extremely small. In addition, in the production of E. coli as a fusion protein inclusion body, in order to obtain mature MTG, there was a problem that the fusion portion had to be cleaved using a restriction protease after expression. Furthermore, since MTG is calcium-independent, it has been found that when the active form of MTG is expressed in the cells of a microorganism, the present enzyme acts on intracellular proteins, which is lethal.

[0009] Therefore, in order to industrially use MTG produced by genetic recombination, it is desired to increase the production of mature MTG having no fusion portion.
The present invention has been made in view of the above, and an object of the present invention is to produce MTG in large quantities using a microorganism such as E. coli. When MTG is expressed using the recombinant DNA of the present invention, a methionine residue is added to the N-terminus of MTG. The addition of a methionine residue to the N-terminus of MTG may pose a problem in terms of safety, such as imparting antigenicity to MTG. From the viewpoint of industrial application, it is also an object of the present invention to produce MTG in which the methionine residue corresponding to the start codon has been removed.

[0010]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors changed the codons to be used for E. coli, preferably by using a multicopy vector (pUC19) and a strong promoter (trp promoter). ), A large-scale expression system for a protein having transglutaminase activity was constructed. In addition, since MTG is expressed and secreted in actinomycetes as a prepro-form, its N-terminus does not contain a methionine residue corresponding to an initiation codon. A methionine residue was added to the N-terminus. In order to solve this problem, the present inventors focused on the substrate specificity of E. coli methionine aminopeptidase, and expressed it by removing the aspartic acid residue which is the N-terminal amino acid of MTG. Thus, a protein having transglutaminase activity in a form from which the N-terminal methionine was removed was obtained, and the present invention was achieved.

That is, the present invention relates to the amino acid sequence shown in SEQ ID NO: 1 from the second serine residue to 33
A protein having a transglutaminase activity containing a sequence leading to the first proline residue, wherein the N-terminal amino acid of the protein corresponds to the second serine in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. It is a protein having a certain transglutaminase activity. The present invention also provides the protein comprising a sequence ranging from the second serine residue to the 331st proline residue in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. The present invention also provides a DNA encoding the protein. The present invention provides a recombinant DNA having the above DNA. Specifically, it is a recombinant DNA that expresses the DNA.
The present invention also provides a transformant transformed by the recombinant DNA.

Further, the present invention provides a method for producing a protein having a transglutaminase activity, which comprises culturing the transformant in a medium, producing a protein having a transglutaminase activity, and recovering the protein.
In addition, the method for producing a protein having transglutaminase activity without an initiating methionine is not limited to removal of the N-terminal aspartic acid in consideration of the substrate specificity of methionine aminopeptidase.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The protein having a transglutaminase activity in the present invention includes SEQ ID NO: 1 in the Sequence Listing.
Starting from the second serine residue of the amino acid sequence described in the above, the sequence up to the 331 th proline residue is essential, and further amino acids may be arranged after the 331 th proline residue. Protein. Among them, those having a sequence from the second serine residue to the 331th proline residue in the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing are preferable. In these amino acid sequences, a protein having a transglutaminase activity, which is a protein in which a certain amino acid is deleted, substituted or inserted, is also included in the present invention. The DNA of the present invention encodes the above protein.

Of these, the fourth A from the N-terminal amino acid
The nucleotide sequence encoding rg is CGT or CGC,
The nucleotide sequence encoding the fifth Val from the N-terminal is GT
DNA that is T or GTA is preferred. Also, the amino acid sequence from the N-terminal amino acid to the fifth: Ser-Asp-
The above-mentioned DNA in which the base sequence encoding Asp-Arg-Val is as follows is preferable. Ser: TCT or TCC Asp: GAC or GAT Asp: GAC or GAT Arg: CGT or CGC Val: GTT or GTA

Of these, the amino acid sequence from the N-terminal amino acid to the fifth amino acid: Ser-Asp-Asp-Arg
The nucleotide sequence encoding -Val is TCT-GAC-GA
DNA that is T-CGT-GTT is preferred. further,
Amino acid sequence from the N-terminal amino acid to the 6th to 9th amino acids: DNA having the following base sequence encoding Thr-Pro-Pro-Ala is preferable. Thr: ACT or ACC Pro: CCA or CCG Pro: CCA or CCG Ala: GCT or GCA

In the present invention, a DNA containing a sequence from the 4th thymine base to the 993th guanine base in the base sequence shown in SEQ ID NO: 2 in the Sequence Listing is preferable. Among them, a DNA consisting of a sequence from the 4th thymine base to the 993th guanine base in the base sequence described in SEQ ID NO: 2 in the Sequence Listing is preferable. In the above DNA sequence, a DNA sequence in which a nucleic acid encoding a certain amino acid has been deleted, a nucleic acid encoding a certain amino acid has been substituted or inserted, and the DNA sequence encodes a protein having transglutaminase activity, is also included in the present invention. include. The recombinant DNA of the present invention has any of the above DNA sequences, and is particularly a recombinant DNA that expresses the above DNA. Of these, trp, tac, lac, t
Recombinant DNA having a promoter selected from the group consisting of rc, λPL and T7 is preferred.

The transformant of the present invention comprises the above recombinant DN
A is a transformant transformed by A. this house,
A transformant transformed using a multicopy vector is preferable, and a transformant belonging to Escherichia coli is preferable. The method for producing a protein having a transglutaminase activity of the present invention comprises culturing the transformant in a medium to produce a protein having a transglutaminase activity.
It is characterized in that it is collected.

Hereinafter, the present invention will be described in detail. (1) It has been known that when MTG is expressed in the cells of microorganisms, it is lethal. However, E. coli
It has been known that when highly expressed in such microorganisms, the expressed protein often becomes an inactive insoluble protein inclusion body. Therefore, a device was devised to highly express MTG as an inactive insoluble protein in E. coli. The structural gene of MTG used for realizing high expression is 99-position from the fourth thymine base in the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.
It is a DNA containing a sequence up to the third guanine base. In consideration of the degeneracy of genetic codons, DNA encoding a protein having the same amino acid sequence may include various nucleotide sequences, but the DNA in the region encoding the N-terminal portion may be used to eliminate formation of higher-order structure of mRNA. The third letter of the degenerate codon was converted to adenine- and uracil-rich codons, and the rest used codons commonly used in E. coli.

As a promoter for expressing the MTG structural gene, a strong promoter usually used for producing a heterologous protein was used, and a terminator was inserted downstream of the MTG structural gene. For example, trp, tac, lac, tr
c, λPL, T7 and other promoters, and trpA, lpp, T4 and other terminators. In addition, in order to improve the efficiency of translation, the type and number of the SD sequence, and the base composition, sequence and length of the region between the SD sequence and the start codon were optimized for MTG gene expression. The region from the promoter to the terminator required for MTG expression can be prepared by a conventionally known chemical synthesis method or the like. An example of this nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing. Although there is an aspartic acid residue after the initiation codon in the amino acid sequence described as SEQ ID NO: 3, it is preferable that this aspartic acid residue be removed as described later.

[0020] The present invention also provides a recombinant DNA that can be used for the expression of MTG. Such a recombinant DNA is obtained by adding a DNA containing the MTG structural gene to a known expression vector corresponding to a desired expression system.
Can be prepared by insertion using a conventionally known method. Here, the expression vector to be used is desirably multi-copy. Known expression vectors that can be used for preparing the recombinant DNA of the present invention include pU
C19, pHSG299 and the like. pUC19 contains the DN of the present invention.
One specific example of the recombinant DNA of the present invention obtained by incorporating A is pUCTRPMTG-02 (+). Furthermore, the present invention relates to various transformants obtained by introducing the recombinant DNA.

The cells which can be the transformant include E. coli.
And so on. As a specific example of E. coli, the JM109 strain (recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, △ (l
ac-proAB) / F '[traD36, proAB +, lacIq, lacZ △ M15])
There is. A protein having transglutaminase activity is produced by culturing such a transformant, for example, a transformant obtained by transforming the E. coli JM109 strain with the vector pUCTRPMTG-02 (+) of the present invention. You.
As the production medium, in addition to the 2xYT medium described in the examples,
A medium usually used for culturing E. coli, such as an LB medium and an M9-casamino acid medium, may be used. In addition, culture conditions,
The conditions for inducing production are appropriately selected according to the type of the vector, promoter host bacteria and the like used. For example, in a recombinant using the trp promoter, an agent such as 3-β-indoleacrylic acid may be added to make the promoter work efficiently. In addition, components such as glucose and casamino acid can be added as needed during the culture. Furthermore, in order to selectively grow the recombinant E. coli, a drug (ampicillin) or the like exhibiting resistance may be added according to the gene such as drug resistance retained in the plasmid.

When the protein having transglutaminase activity produced by the above method is extracted from a cultured strain, the cells are collected after culturing, the cells are suspended in a buffer, treated with lysozyme, freeze-thawed, and sonicated. After processing such as crushing, the cells are disrupted and then separated into a supernatant and a precipitate by centrifugation.
Since the protein having the transglutaminase activity is produced as a protein inclusion body and separated into a precipitate, the protein can be solubilized with a denaturing agent or the like, and then the denaturing agent can be removed, followed by separation and purification. As a denaturant for solubilizing the produced protein inclusion body, urea (for example, 8M), guanidine hydrochloride (for example, 6M) and the like can be mentioned. When these denaturing agents are removed by dialysis or the like, the protein is regenerated as a protein having transglutaminase activity. As the dialysis solution used for dialysis, a phosphate buffer or a Tris-HCl buffer may be used. In addition, as a method for removing the denaturing agent, there are a dialysis method, a dilution method, an ultrafiltration method and the like, and regeneration of the activity can be expected by using any of them.

Further, after the activity is regenerated, the active protein can be separated and purified by appropriately combining known separation and purification methods. For example, salting out, dialysis, ultrafiltration,
Gel filtration, SDS-polyacrylamide electrophoresis,
Examples include ion exchange chromatography, affinity chromatography, reversed-phase high-performance liquid chromatography, and isoelectric focusing. (2) The present invention is also a protein having a transglutaminase activity, having a sequence from the second serine residue to the 331st proline residue in the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing. Analysis of the N-terminus of MTG produced by the transformant transformed with the recombinant DNA having the DNA described in SEQ ID NO: 3 in the Sequence Listing shows that
It was found that a (formyl) methionine residue of the initiation codon remained in most of them.

In general, when a gene encoding a foreign protein is expressed in E. coli, the target protein is designed to be located after a methionine residue encoded by ATG which is a translation initiation signal of the gene. . In the case of a natural protein, the N-terminal methionine residue of a protein obtained as a result of translation from a gene is known to be efficiently cleaved by methionine aminopeptidase. Residues are not always truncated. It has been found that the substrate specificity of methionine aminopeptidase depends on the type of amino acid residue located next to the methionine residue. When the amino acid residue located next to the methionine residue is an alanine residue, glycine residue, serine residue, etc., the methionine residue is easily cleaved, but aspartic acid, asparagine, lysine, arginine, leucine, etc. Is difficult to cut (Nature 326 315 (198
7)).

The N-terminal amino acid residue of MTG is an aspartic acid residue. If a methionine residue derived from the initiation codon is located immediately before the aspartic acid residue, the sequence is unlikely to act on methionine aminopeptidase.
The N-terminal methionine residue often remains without being removed. However, since there is a serine residue next to the N-terminal aspartic acid of MTG, by deleting the aspartic acid residue, the amino acid residue located next to the methionine residue derived from the initiation codon can be By designing the sequence to be a serine residue, which is an amino acid residue on which methionine aminopeptidase easily acts, a protein having transglutaminase activity in which the N-terminal methionine residue is cleaved can be efficiently produced.

Although a recombinant protein having one amino acid residue shorter than that of natural MTG is obtained, the function of this recombinant protein is not different from that of natural MTG. That is, MTG activity is not lost due to the deletion of one amino acid. In addition, a protein having transglutaminase activity in which a methionine residue is not cleaved may newly acquire antigenicity, whereas a sequence shortened by a few residues may have a new antigenicity without natural MTG. There is no problem in terms of safety because it is not considered to be given to the public. In fact, microbial transglutaminase (MT
G) Met-Se in which the aspartic acid residue at the N-terminus has been removed
Design the sequence r-Asp-Asp-Arg -...
When produced in E. coli, methionine residues were efficiently removed, and a protein having the sequence of Ser-Asp-Asp-Arg -... was obtained. It was also confirmed that the specific activity of the obtained protein was not different from that of the native MTG.

A method for producing a protein having a transglutaminase activity and having a sequence from the second serine residue to the 331th proline residue in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing is as follows. . That is, as the MTG structural gene present on the recombinant DNA which can be used for MTG expression described in the above (1), the MTG structural gene from the second serine residue of the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing is used. A DNA encoding a protein having a transglutaminase activity having a sequence up to the proline residue at position 331 is employed. Specifically, a DNA having a sequence from the 4th thymine base to the 993th guanine base in the base sequence described in SEQ ID NO: 2 in the Sequence Listing is employed.

Examples of the method for modifying the N-terminal sequence include, in addition to the conventional recombinant DNA technology, a site-directed mutagenesis technique, a method using PCR for the full length or a part of the MTG gene, and a method in which the sequence of the modified portion is treated with a restriction enzyme. For exchanging with a synthetic DNA fragment. The transformant transformed with the recombinant DNA thus obtained is cultured in a medium to produce a protein having a transglutaminase activity, which is recovered. The method for preparing the transformant and the method for producing the protein are the same as described above. Since the produced protein has the sequence of Met-Ser -..., which is susceptible to cleavage of methionine residues by methionine aminopeptidase, methionine residues are cleaved in E. coli cells and start from serine residues. It is a protein.

MTG having a methionine residue added to the N-terminus
Have not been found in nature, but the present inventors have found that some of the natural MTGs have an aspartic acid residue removed and the N-terminus is serine. From this, it can be said that those having a methionine residue at the N-terminus are proteins having a sequence different from that of natural MTG, whereas those having a serine residue at the N-terminus are included in the sequence of natural MTG. Since there is actually one having such a sequence, it can be said that it is equivalent to natural MTG.
That is, the presence or absence of the antigenicity of a protein is very important
In the case of enzymes used in foods such as TG, particularly, to produce a protein having a transglutaminase activity having a sequence equivalent to that of natural MTG, that is, to produce a sequence in which the N-terminal methionine residue is cleaved. is important. Hereinafter, examples of the present invention will be described, but the present invention should not be limited to these.

[0030]

【Example】

Mass expression of MTG in E. coli <1> Construction of MTG expression plasmid pTRPMTG-01 The MTG gene has already been fully synthesized in consideration of the codon usage of E. coli and yeast (Japanese Patent Laid-Open No. 5-199883). ). However, this gene sequence was not optimal for expression in E. coli. That is, the codons of 30 arginine residues are all minor codons A
GA. Therefore, first, about 200 bases from the N-terminus of the MTG gene were resynthesized so as to be optimal for the expression of E. coli.

The promoter that transcribes the MTG gene is
A trp promoter, whose transcription is easily induced by lack of tryptophan in the medium, was used. Plasmid pTTG2-22 (JP-A-6-225775), which highly expresses the red sea bream transglutaminase (TG) gene, uses the trp promoter, and the sequence upstream of the red sea bream TG gene is E.co.
li is designed to highly express heterologous proteins. For construction of pTRPMTG-01, as shown in FIG. 1, the trtr of the red sea bream TG expression plasmid pTTG2-22 (JP-A-6-225775) was used.
From the ClaI site downstream of the p promoter to the BglII site downstream, the synthetic DNA gene ClaI / HpaI fragment and pGEM15BTG
It was constructed by replacing the HpaI / BamHI fragment (small) of (JP-A-6-30771).

The ClaI / HpaI fragment of the synthetic DNA gene was
2-22 shows a sequence from the ClaI site downstream of the trp promoter to the translation initiation codon and a gene sequence having 216 bases from the N-terminus of the MTG gene. The nucleotide sequence of the MTG structural gene portion was determined with reference to the codon usage of E. coli so as to optimize the expression of E. coli. However, mRNA
In order to eliminate the formation of higher-order structures, the third character of the degenerate codon in the region encoding the N-terminal portion was converted to a codon rich in adenine and uracil, and care was taken not to continue the same base as much as possible. The synthetic DNA gene ClaI / HpaI fragment was designed to have EcoRI and HindIII at its ends. Divide the designed gene into about 40 to 50 bases so that the + and-strands overlap each other, corresponding to each sequence
Twelve DNAs were synthesized (SEQ ID NOS: 4 to 15 in the Sequence Listing). The 5 'end of this synthetic DNA was phosphorylated, and the paired synthetic DNAs were annealed and ligated. Thereafter, acrylamide gel electrophoresis was performed to cut out DNA of the desired size, and the DNA was inserted into the EcoRI / HindIII site of pUC19.
The nucleotide sequence was confirmed, and the correct one was named pUCN216.
From this pUCN216, a ClaI / HpaI fragment (small) was cut out.
It was used for construction of pTRPMTG-01.

<2> Construction of MTG Expression Plasmid pTRPMTG-02 Since E. coli JM109 carrying pTRPMTG-01 did not express MTG at a high level, a portion (777 bases) other than the N-terminal modified portion of the MTG gene was used. ) Was also modified for E. coli. Since it is difficult to synthesize 777 bases at once, E.co.
After determining the nucleotide sequence with reference to the codon usage of li, four blocks (B1, 2, 3,
4) It was decided to synthesize them separately. Each block was designed to have EcoRI and HindIII at the ends. Divide the designed gene into about 40 to 50 bases so that the + and-strands overlap each other, corresponding to each sequence
A total of 40 DNAs were synthesized, 10 for each block (SEQ ID NOS: 16 to 55 in the Sequence Listing). The 5 'end of this synthetic DNA was phosphorylated, and the paired synthetic DNAs were annealed and ligated. Thereafter, acrylamide gel electrophoresis was performed to cut out DNA of the desired size, and EcoRI / HindII of pUC19 was used.
I was incorporated into the site. The nucleotide sequence was confirmed, and the correct ones were named pUCB1, B2, B3, and B4, respectively. Next, as shown in FIG. 2, after connecting B1 and B2 and B3 and B4, pTRPMT
By replacing the corresponding part of G-01, pTRPMTG-02 was constructed. pT
The nucleotide sequence of the highly expressed MTG gene present on RPMTG-02 is shown in SEQ ID NO: 3 in the Sequence Listing.

<3> MTG expression plasmid pUCTRPMTG-02
Construction of (+) and (−) E. coli JM109 carrying this pTRPMTG-02 also did not highly express MTG, so it was decided to copy the plasmid into multiple copies. EcoO109I containing trp promoter of pTRPMTG-02
After blunting the fragment (small), the multicopy plasmid pUC1
9 at the HincII site, lacZ promoter and trp
A promoter having the same promoter (pUCTRPMTG-02 (+)) and a promoter having the opposite orientation (pUCTRPMTG-02 (-)) were constructed. <4> Expression of MTG E. coli JM1 transformed with pUCTRPMTG-02 (+) and pUC19
09 with 3 ml of 2xYT containing 150 μg / ml ampicillin
The medium was shake-cultured at 37 ° C for 10 hours (preculture). 0.5 ml of this preculture was added to 50 μg / ml ampicillin
The mixture was added to 2 ml of 2xYT medium and cultured with shaking at 37 ° C for 20 hours.

The cells were collected from the culture solution and sonicated. FIG. 3 shows the results of developing all fractions, centrifuged supernatant fractions, and centrifuged precipitate fractions of the cell lysate by SDS-polyacrylamide electrophoresis. High expression of a protein having the same molecular weight as MTG was confirmed in all fractions of disrupted bacterial cells of pUCTRPMTG-02 (+) / JM109 and in the centrifuged precipitate fraction. In addition, it was confirmed by Western blotting that this protein reacted with a mouse anti-MTG antibody. The expression level of this protein is 50
It was 0 to 600 mg / L. In addition, 3-β-
Sufficiently high expression was observed without adding indoleacrylic acid. Furthermore, when Western blotting was performed using a mouse anti-MTG antibody, only a small amount of MTG was expressed in the centrifuged supernatant fraction, and almost all of the expressed MTG was insoluble protein inclusion bodies. Do you get it.

<5> Construction of MTG Expression Plasmid pTRPMTG-00 Next, in order to show that the codon change of the MTG gene led to a significant increase in the expression level, the MTG gene of pTRPMTG-02 was previously completely synthesized. Gene sequence (Japanese Unexamined Patent Publication No. 6-30771)
PTRPMTG-00 was constructed. For construction of pTRPMTG-00, as shown in FIG.
PvuII / PstI fragment (small) and PstI / H cut out from RPMTG-02
It was constructed by ligating an imdIII fragment (small, including a PvuII site) and a PvuII / HindIII fragment (small) of pGEM15BTG (JP-A-6-30771). <6> MTG expression plasmid pUCTRPMTG-00 (+),
Construction of (−) Further, pTRPMTG-00 was made into multiple copies. pTRPMTG-00 tr
After blunting the EcoO109I fragment (small) containing the p promoter and the trpA terminator, the multicopy plasmid pUC19
The lacZ promoter and the trp promoter were identical (pUCTRPMTG-00 (+)) and inverted (pUCTRPMTG-00 (-)).

<7> Comparison of expression levels of MTG pUCTRPMTG-02 (+), (−), pUCTRPMTG-00 (+),
(-), PTRPMTG-02, pTRPMTG-01, pTRPMTG-00, pUC19
E. coli JM109 transformed in 3) was cultured with shaking in 3 ml of 2 × YT medium containing 150 μg / ml of ampicillin at 37 ° C. for 10 hours (preculture). Add 0.5 ml of this preculture to 150 μg /
Add to 50 ml of 2xYT medium containing ml of ampicillin,
The cells were cultured with shaking at 37 ° C for 20 hours. Table 1 shows the results of collecting cells from the culture solution and measuring the expression level of MTG. It was found that newly constructed E. coli having pTRPMTG-00, pUCTRPMTG-00 (+), and (-) did not highly express MTG. From these results, it was found that high expression of MTG requires both changing the codon of the MTG gene for E. coli and increasing the number of copies of the plasmid.

[0038]

[Table 1] Table-1 Strain name MTG expression level pUCTRPMTG-02 (+) / JM109 ++++ pUCTRPMTG-02 (-) / JM109 ++++ pUCTRPMTG-00 (+) / JM109 + pUCTRPMTG-00 (-) / JM109 + pTRPMTG-02 / JM109 + pTRPMTG-01 / JM109 + pTRPMTG-00 / JM109 − pUC19 / JM109 − +++: 300 mg / l or more +: 5 mg / l or less −: Not expressed

<8> Analysis of N-terminal Amino Acid of Expressed MTG When the N-terminal amino acid residue of the expressed MTG protein inclusion body was analyzed, about 60% of the N-terminal sequence was methionine residue and about 40%. Was a formyl methionine residue. In order to remove the (formyl) methionine residue corresponding to the start codon, the following contrivance was made. <9> Deletion of N-terminal aspartic acid residue of MTG The base sequence corresponding to the aspartic acid residue was deleted by performing PCR using pUCN216 containing 216 bases corresponding to the N-terminal of MTG as a template. . pUCN216 is MT
About 216 bp including the N-terminal ClaI-HpaI fragment of G
Plasmid cloned into the oRI / HindIII site. pF01 (SEQ ID NO: 56) and pR01 (SEQ ID NO: 57) are primers having a sequence in a vector,
pDELD (SEQ ID NO: 58) is a deletion of the nucleotide sequence corresponding to the Asp residue, pHd01 (SEQ ID NO: 5).
Fig. 9) shows the transformation of C to G to crush the HindIII site. pF01 and pDELD are sense primers, and pR01 and pHd01 are antisense primers.

First, PCR was performed at 94 ° C. for 30 minutes at 94 ° C. on pUCN216 using a combination of pF01 and pHd01, and pDELD and pR01.
35 cycles were performed under the conditions of 1 second at 55 ° C. and 2 minutes at 72 ° C. After each PCR product was extracted with phenol / chloroform, ethanol precipitation was carried out and dissolved in 100 μL of H2O. Mix 1 μL of each PCR product, heat denature at 94 ° C for 10 minutes, and combine the primers with pF01 and pHd01 to perform PCR for 30 seconds at 94 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C. For 35 cycles. Phenol /
After extraction with chloroform, ethanol precipitated
ndIII, EcoRI treatment, pUC19 subcloning and pU
CN216D was obtained (FIG. 5). When the sequence of the obtained pUCN216D was confirmed, it was as intended.

<10> Construction of Plasmid Deleting Aspartic Acid Residue The Ecoo109I / HpaI fragment (small) of pUCN216D was replaced with pUCB1-2 (MT
A plasmid in which the HpaI / BglII fragment of the G gene was cloned into the EcoRI / HindIII site of pUC19)
It was ligated with the fragment (large) to obtain pUCNB1-2D. Furthermore, pUCNB1-2
The ClaI / BglII fragment (small) of D was ligated to the MTG high expression plasmid pUCTRPMTG-02 (+) ClaI / BglII fragment (large), and the aspartic acid residue-removed MTG expression plasmid pUCT
RPMTG (+) D2 (FIG. 6). As a result, as shown in FIG. 7, MT lacking GAT corresponding to an aspartic acid residue was obtained.
A plasmid containing the G gene was obtained. <11> Expression of MTG lacking aspartic acid residue E. coli JM109 transformed with pUCTRPMTG (+) D2 was
In 3 ml of 2 × YT medium containing μg / ml ampicillin, 3
The cells were cultured with shaking at 7 ° C for 10 hours (pre-culture). This preculture
0.5 ml to 50 ml of 2xY containing 150 μg / ml ampicillin
The mixture was added to T medium, and cultured with shaking at 37 ° C for 20 hours. From the results of Coomassie brilliant blue staining after SDS-polyacrylamide gel electrophoresis and Western blotting using a mouse anti-MTG antibody, the disrupted supernatant and precipitate after sonication of the cells, MTG protein lacking aspartic acid residues was detected in the precipitate, that is, in the insoluble fraction. This indicates that the aspartic acid residue-deleted MTG protein was accumulated in the cells as a protein inclusion body.

When the N-terminal amino acid sequence of this protein inclusion body was analyzed, as shown in FIG.
About 90% of the N-terminal sequence was serine. <8> and <1
Comparison of the results for the N-terminal amino acid of the expressed MTG obtained in 1) is as shown in Table 2. The deletion of the N-terminal aspartic acid residue of the MTG resulted in the expression of the expressed MTG.
It was found that the starting methionine added to the N-terminal of G was efficiently removed.

[0043]

[Table 2] Table-2 Strain name Terminal amino acid f-Met Met Asp Ser pUCTRPMTG-02 (+) / JM109 40% 60% ND- pUCTRPMTG (+) D2 / JM109 ND 10%-90%

<12> Solubilization of Aspartic Acid Residue Deleted MTG Inclusion Body, Regeneration of Activity, and Measurement of Specific Activity The aspartic acid deletion MTG inclusion body was partially purified by centrifugation several times, and then 2 mg / ml. 8M urea (50mM
After dissolving in phosphate buffer (pH 5.5)), remove the precipitate by centrifugation, dilute with 50 mM phosphate buffer (pH 5.5) to 0.5 M urea, and further dialyze against 50 mM phosphate buffer (pH 5.5). Do urea except. When this was applied to a MonoS column,
The peak having TG activity eluted when the NaCl concentration was 100-150 mM. The specific activity of this fraction was measured by the hydroxamate method.
The specific activity of TG was about 30 U / mg. This is the natural MT
The specific activity was equivalent to that of G, and it was revealed that the deletion of the aspartic acid residue did not affect the specific activity.

[0045]

[Sequence list]

SEQ ID NO: 1 Sequence length: 331 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met 1 5 10 15 Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn 20 25 30 Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg 35 40 45 Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys 50 55 60 Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu 65 70 75 80 Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn 85 90 95 Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val 100 105 110 Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu 115 120 125 Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser 130 135 140 Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala 145 150 155 160 Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn 165 170 175 Thr Pro Ser Phe Lys G lu Arg Asn Gly Gly Asn His Asp Pro Ser Arg 180 185 190 Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg 195 200 205 Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210 215 220 Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile 225 230 235 240 Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr 245 250 255 Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp 260 265 270 Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met 275 280 285 His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp 290 295 300 Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 325 330

SEQ ID NO: 2 Sequence length: 993 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: CDS location : 1..993 Method for determining characteristics: S sequence GAT TCT GAC GAT CGT GTT ACT CCA CCA GCT GAA CCA CTG GAT CGT ATG 48 Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met 1 5 10 15 CCA GAT CCA TAT CGT CCA TCT TAT GGT CGT GCT GAA ACT GTT GTT AAT 96 Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn 20 25 30 AAT TAT ATT CGT AAA TGG CAA CAA GTT TAT TCT CAT CGT GAT GGT CGT 144 Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg 35 40 45 AAA CAA CAA ATG ACT GAA GAA CAA CGT GAA TGG CTG TCT TAT GGT TGC 192 Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys 50 55 60 GTT GGT GTT ACT TGG GTT AAC TCT GGT CAG TAT CCG ACT AAC CGT CTG 240 Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu 65 70 75 80 GCA TTC GCT TCC TTC GAT GAA GAT CGT TTC AAG AAC GAA CTG AAG AAC 288 Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn 85 90 95 GGT CGT CCG CGT TCT GGT GAA ACT CGT GCT GAA TTC GAA GGT CGT GTT 336 Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val 100 105 110 GCT AAG GAA TCC TTC GAT GAA GAG AAA GGC TTC CAG CGT GCT CGT GAA 384 Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu 115 120 125 GTT GCT TCT GTT ATG AAC CGT GCT CTA GAG AAC GCT CAT GAT GAA TCT 432 Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser 130 135 140 GCT TAC CTG GAT AAC CTG AAG AAG GAA CTG GCT AAC GGT AAC GAT GCT 480 Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala 145 150 155 160 CTG CGT AAC GAA GAT GCT CGT TCT CCG TTC TAC TCT GCT CTG CGT AAC 528 Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn 165 170 175 ACT CCG TCC TTC AAA GAA CGT AAC GGT GGT AAC CAT GAT CCG TCT CGT 576 Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg 180 185 190 ATG AA A GCT GTT ATC TAC TCT AAA CAT TTC TGG TCT GGT CAG GAT AGA 624 Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg 195 200 205 TCT TCT TCT GCT GAT AAA CGT AAA TAC GGT GAT CCG GAT GCA TTC CGT 672 Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210 215 220 CCG GCT CCG GGT ACT GGT CTG GTA GAC ATG TCT CGT GAT CGT AAC ATC 720 Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile 225 230 235 240 CCG CGT TCT CCG ACT TCT CCG GGT GAA GGC TTC GTT AAC TTC GAT TAC 768 Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr 245 250 255 GGT TGG TTC GGT GCT CAG ACT GAA GCT GAT GCT GAT AAG ACT GTA TGG 816 Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp 260 265 270 270 ACC CAT GGT AAC CAT TAC CAT GCT CCG AAC GGT TCT CTG GGT GCT ATG 864 Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met 275 280 285 CAT GTA TAC GAA TCT AAA TTC CGT AAC TGG TCT GAA GGT TAC TCT GAC 912 His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp 290 29 5 300 TTC GAT CGT GGT GCT TAC GTT ATC ACC TTC ATT CCG AAA TCT TGG AAC 960 Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 ACT GCT CCG GAC AAA GTT AAA CAG GGT TGG CCG 993 Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 325 330

SEQ ID NO: 3 Sequence length: 1518 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: CDS location : 87..1082 Method for determining characteristics: S sequence TTCCCCTGTT GACAATTAAT CATCGAACTA GTTAACTAGT ACGCAAGTTC ACGTAAAAAG 60 GGTATCGATT AGTAAGGAGG TTTAAA ATG GAT TCT GAC GAT CGT GTT ACT CCA 113 Met Asp Ser Asp Asp ArgCATG CAT 5 CGT ATG CCA GAT CCA TAT CGT CCA TCT TAT 161 Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser Tyr 10 15 20 25 GGT CGT GCT GAA ACT GTT GTT AAT AAT TAT ATT CGT AAA TGG CAA CAA 209 Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln 30 35 40 GTT TAT TCT CAT CGT GAT GGT CGT AAA CAA CAA ATG ACT GAA GAA CAA 257 Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln 45 50 55 CGT GAA TGG CTG TCT TAT GGT TGC GTT GGT GTT ACT TGG GTT AAC TCT 305 Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser 60 65 70 GGT CAG TAT CCG ACT AAC CGT CTG GCA TTC GCT TCC TTC GAT GAA GAT 353 Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser Phe Asp Glu Asp 75 80 85 CGT TTC AAG AAC GAA CTG AAG AAC GGT CGT CCG CGT TCT GGT GAA ACT 401 Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr 90 95 100 105 CGT GCT GAA TTC GAA GGT CGT GTT GCT AAG GAA TCC TTC GAT GAA GAG 449 Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser Phe Asp Glu Glu 110 115 120 AAA GGC TTC CAG CGT GCT CGT GAA GTT GCT TCT GTT ATG AAC CGT GCT 497 Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val Met Asn Arg Ala 125 130 135 CTA GAG AAC GCT CAT GAT GAA TCT GCT TAC CTG GAT AAC CTG AAG AAG 545 Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp Asn Leu Lys Lys 140 145 150 GAA CTG GCT AAC GGT AAC GAT GCT CTG CGT AAC GAA GAT GCT CGT TCT 593 Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu Asp Ala Arg Ser 155 160 165 CCG TTC TAC TCT GCT CTG CGT AAC ACT CCG TCC TTC AAA GAA CGT AAC 641 Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser Phe Lys Glu Arg Asn 170 175 180 185 GGT GGT AAC CAT GAT CCG TCT CGT ATG AAA GCT GTT ATC TAC TCT AAA 689 Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala Val Ile Tyr Ser Lys 190 195 200 CAT TTC TGG TCT GGT CAG GAT AGA TCT TCT TCT GCT GAT AAA CGT AAA 737 His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser Ala Asp Lys Arg Lys 205 210 215 TAC GGT GAT CCG GAT GCA TTC CGT CCG GCT CCG GGT ACT GGT CTG GTA 785 Tyr Gly Asp Pro Asp Ala Phe Arg Pro Ala Pro Gly Thr Gly Leu Val 220 225 230 GAC ATG TCT CGT GAT CGT AAC ATC CCG CGT TCT CCG ACT TCT CCG GGT 833 Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro Gly 235 240 245 GAA GGC TTC GTT AAC TTC GAT TAC GGT TGG TTC GGT GCT CAG ACT GAA 881 Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu 250 255 260 265 GCT GAT GCT GAT AAG ACT GTA TGG ACC CAT GGT AAC CAT TAC CAT GCT 929 Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly Asn His Tyr His Ala 270 275 280 CCG AAC GGT TCT CTG GGT GCT ATG CAT GTA TAC GAA TCT AAA TTC CGT 977 Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe Arg 285 290 295 AAC TGG TCT GAA GGT TAC TCT GAC TTC GAT CGT GGT GCT TAC GTT ATC 1025 Asn Trp Ser Glu Gly Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val Ile 300 305 310 ACC TTC ATT CCG AAA TCT TGG AAC ACT GCT CCG GAC AAA GTT AAA CAG 1073 Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro Asp Lys Val Lys Gln 315 320 325 GGT TGG CCG TAATGAAAGC TTGGATCTCT AATTACTGGA CTTCACACAG ACTAAAATAG TTT AGATTGA TATTATATTAATTG TTTCCTAGAT GTGAGGTGGA GGTGATGTAT 1191 AAGGTAGATG ATGATCCTCT ACGCCGGACG CATCGTGGCC GGCATCACCG GCGCCACAGG 1251 TGCGGTTGCT GGCGCCTATA TCGCCGACAT CACCGATGGG GAAGATCGGG CTCGCCACTT 1311 CGGGCTCATG AGCGCTTGTT TCGGCGTGGG TATGGTGGCA GGCCCCGTGG CCGGGGGACT 1371 GTTGGGCGCC ATCTCCTTGC ATGCACCATT CCTTGCGGCG GCGGTGCTCA ACGGCCTCAA 1431 CCTACTACTG GGCTGCTTCC TAATGCAGGA GTCGCATAAG GGAGAGCGTC GAGAGCCCGC 1491 CTAATGAGCG GGCTTTTTTT TCAGCTG 1518

SEQ ID NO: 4 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 5 Sequence length: 41 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 6 Sequence length: 41 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 7 Sequence length: 41 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 8 Sequence length: 41 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 9 Sequence length: 41 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 10 Sequence length: 41 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 11 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 12 Sequence length: 41 Sequence type: Number of nucleic acid chains: Single-stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 13 Sequence length: 41 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 14 Sequence length: 42 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 15 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 16 Sequence length: 38 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 17 Sequence length: 41 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 18 Sequence length: 49 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GCATTCGCTT CCTTCGATGA AGATCGTTTC AAGAACGAAC TGAAGAACG 49

SEQ ID NO: 19 Sequence length: 49 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GGACGACCGT TCTTCAGTTC GTTCTTGAAA CGATCTTCAT CGAAGGAAG 49

SEQ ID NO: 20 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 21 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 22 Sequence length: 48 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GAAGGTCGTG TTGCTAAGGA ATCCTTCGAT GAAGAGAAAG GCTTCCAG 48

SEQ ID NO: 23 Sequence length: 48 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GAGCACGCTG GAAGCCTTTC TCTTCATCGA AGGATTCCTT AGCAACAC 48

SEQ ID NO: 24 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 25 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 26 Sequence length: 45 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 27 Sequence length: 50 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTTCTTCAGG TTATCCAGGT AAGCAGATTC ATCATGAGCG TTCTCTAGAG 50

SEQ ID NO: 28 Sequence length: 49 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTGAAGAAGG AACTGGCTAA CGGTAACGAT GCTCTGCGTA ACGAAGATG 49

SEQ ID NO: 29 Sequence length: 49 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GAGAACGAGC ATCTTCGTTA CGCAGAGCAT CGTTACCGTT AGCCAGTTC 49

SEQ ID NO: 30 Sequence length: 40 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 31 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 32 Sequence length: 47 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTTCAAAGAA CGTAACGGTG GTAACCATGA TCCGTCTCGT ATGAAAG 47

SEQ ID NO: 33 Sequence length: 47 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GATAACAGCT TTCATACGAG ACGGATCATG GTTACCACCG TTACGTT 47

SEQ ID NO: 34 Sequence length: 45 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 35 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 36 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 37 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 38 Sequence length: 48 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CGGATGCATT CCGTCCGGCT CCGGGTACTG GTCTGGTAGA CATGTCTC 48

SEQ ID NO: 39 Sequence length: 48 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence GATCACGAGA CATGTCTACC AGACCAGTAC CCGGAGCCGG ACGGAATG 48

SEQ ID NO: 40 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 41 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 42 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 43 Sequence length: 40 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 44 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 45 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 46 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 47 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 48 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 49 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 50 Sequence length: 37 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 51 Sequence length: 37 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 52 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 53 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 54 Sequence length: 38 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 55 Sequence length: 34 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA

SEQ ID NO: 56 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

SEQ ID NO: 57 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 58 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 59 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a diagram showing the construction of an MTG expression plasmid pTRPMTG-01.

FIG. 2 is a construction diagram of an MTG expression plasmid pTRPMTG-02.

FIG. 3 is a developed view of SDS-polyacrylamide electrophoresis showing that MTG was expressed.

FIG. 4 is a construction diagram of an MTG expression plasmid pTRPMTG-00.

FIG. 5 is a construction diagram of plasmid pUCN216D.

FIG. 6: MTG expression plasmid pUCTRTMTG
(+) It is a construction diagram of D2.

FIG. 7 shows that GAT corresponding to an aspartic acid residue was deleted.

FIG. 8 shows that the N-terminal amino acid is serine.

──────────────────────────────────────────────────の Continuing on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 9/10 C12R 1:19) (72) Inventor Katsuya Shiguro 1-1 Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Food Research Institute, Inc.

Claims (16)

[Claims] 1. A protein having a transglutaminase activity, comprising a sequence from the second serine residue to the 331st proline residue in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing, wherein the protein has A protein having a transglutaminase activity, wherein the terminal amino acid corresponds to the second serine in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. 2. The protein according to claim 1, comprising a sequence from the second serine residue to the 331st proline residue in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. 3. A DNA encoding the protein according to claim 1 or 2. 4. The nucleotide sequence encoding Arg at the fourth position from the N-terminal amino acid is CGT or CGC, and the nucleotide sequence encoding Val at the fifth position from the N-terminal is GTT or GTC.
The DNA according to claim 3, which is TA. 5. The amino acid sequence from the N-terminal amino acid to the fifth amino acid sequence: The base sequence encoding Ser-Asp-Asp-Arg-Val is as follows:
NA. Ser: TCT or TCC Asp: GAC or GAT Asp: GAC or GAT Arg: CGT or CGC Val: GTT or GTA 6. The amino acid sequence from the N-terminal amino acid to the fifth amino acid: Ser-Asp-Asp-Arg-Val has a nucleotide sequence encoding TCT-GAC-GAT-CGT-
The DNA according to claim 5, which is GTT. 7. The DNA according to claim 5, wherein the nucleotide sequence encoding the amino acid sequence from the N-terminal amino acid to the sixth to ninth amino acids: Thr-Pro-Pro-Ala is as follows. Thr: ACT or ACC Pro: CCA or CCG Pro: CCA or CCG Ala: GCT or GCA 8. A DNA comprising a sequence ranging from the 4th thymine base to the 993th guanine base in the base sequence shown in SEQ ID NO: 2 in the sequence listing. 9. The DNA according to claim 8, comprising a sequence ranging from the 4th thymine base to the 993th guanine base in the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. 10. A recombinant DNA having the DNA according to claim 3. 11. The recombinant DNA according to claim 10, which expresses the DNA according to claim 3 to 9. 12. Trp, tac, lac, trc, λ
12. The recombinant DNA according to claim 10, which has a promoter selected from the group consisting of PL and T7. A transformant transformed with the recombinant DNA according to any one of claims 10 to 12. 14. The transformant according to claim 13, which has been transformed with a multicopy vector. 15. The method according to claim 1, which belongs to Escherichia coli.
15. The transformant according to 3 or 14. 16. A transfectant having transglutaminase activity, wherein the transformant according to any one of claims 13 to 15 is cultured in a medium to produce a protein having transglutaminase activity, and the protein is recovered. A method for producing proteins.