JPH0630771A - Production of bacterial transglutaminase and related material - Google Patents

Abstract

PURPOSE:To mass produce bacterial transglutaminase by using a bacterium such as Escherichia coli. CONSTITUTION:Escherichia coli retaining a plasmid manifesting a fused protein containing bacterial transglutaminase and a hydrophilic peptide existing at the amino end side of the bacterial transglutaminase is cultured to produce a bacterial transglutaminase-fused protein as an inert sealed material in cells. The sealed material is recovered from the cells, the sealed material is solubilized by using a denaturing agent and the denaturing agent is removed to give a fused protein having transglutaminase activity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing bacterial transglutaminase and related substances, and
The present invention relates to a method for producing transglutaminase by efficiently regenerating an active protein from an inactive protein inclusion body produced by Escherichia coli (Escherichia coli).

[0002]

BACKGROUND OF THE INVENTION Transglutaminase is an enzyme that catalyzes the acyl transfer of the γ-carboxamide group of the Gln residue in the peptide chain of a protein. When this enzyme acts on a protein, ε- (γ-Gln) -Lys cross-linking reaction, Gl
Substitution reaction to Glu by deamidation of n may occur.

This transglutaminase is used for producing gelled foods such as jelly, yogurt, cheese, gelled cosmetics, etc., and improving the meat quality of meat (for example, Japanese Patent Publication No. 1-50382). Further, it is an industrially highly applicable enzyme, such as being used for the production of heat-stable microcapsule materials and carriers for immobilized enzymes.

[0004] The primary structure of transglutaminase has so far been known for animal origin and bacteria origin (bacterial transglutaminase: hereinafter referred to as "BTG").

The former is a calcium ion-dependent enzyme, which is distributed in animal organs, skin, blood and the like. Examples include guinea pig liver transglutaminase (K. Ikura et al . Biochiemistry 27 , 2898 (198
8)), human epidermal keratinocyte transglutaminase (MA Phillips et al . Proc . Natl . Acad . Sci . USA)
87 9333 (1990)), human blood coagulation factor XIII (A. Ichino
se et al . Biochemistry 25 6900 (1990)), etc., regarding guinea pig liver transglutaminase,
There are reports of expression production in E. coli (Koji Ikura et al.,
Proceedings of the Japan Society for Agricultural Chemistry, 1988, p62).

Regarding the latter, a calcium ion-independent one has been discovered from a bacterium belonging to the genus Streptoverticillium. An example is Streptoverticillium griseo.
carneum ) IFO 12776, Streptoverticillium, Cinnamonium, Sub-SP, Cinnamonium ( Streptover
ticillium cinnamoneum sub sp. cinnamoneum ) IFO 128
52, Streptoverticillium mobaraens
overticillium mobaraense ) IFO 13819 and the like (JP-A-64-27471).

As a result of peptide mapping and gene structure analysis, the primary structure of transglutaminase produced by these bacteria has been found to have no homology with that of animal origin (European Patent Publication).
0 481 504 A1).

[0008] By the way, since transglutaminase has been conventionally produced from the above animals and fungi,
There was a problem in terms of efficiency. Therefore, the group of the inventors of the present application has attempted, for example, direct expression in Escherichia coli, secretory expression into periplasm, etc. with the aim of producing BTG by a genetic engineering method (European Patent Publication No. 0 481 504). A1).

[0009]

However, as described above, since BTG is calcium ion-independent, when it is produced by a microorganism such as Escherichia coli, this enzyme acts on a protein required for the growth of bacterial cells. As a result, there were problems such as fatality and low production.

Therefore, in order to industrially utilize BTG produced by gene recombination, it is desired to increase the production amount. The present invention has been made from the above viewpoint, and an object thereof is to produce a large amount of BTG using a microorganism such as Escherichia coli.

[0011]

[Means for Solving the Problems] In order to solve the above problems, the present inventors tried to express BTG in large amounts as an inactive protein inclusion body under the control of trc promoter which is one of strong promoters. Attempts were made, but only a slight amount of BTG expression was observed, and the transformant became lethal (see Comparative Example described below).

Therefore, as a result of intensive studies to overcome these problems, as a result of producing and accumulating BTG as an inactive fusion protein inclusion body in Escherichia coli, solubilizing the inclusion body with a protein denaturant, The present inventors have found that a protein having a transglutaminase activity can be mass-produced by regenerating the activity through a dedenaturing agent process, and the present invention has been completed.

That is, the present invention provides a fusion protein containing a bacterial transglutaminase and a hydrophilic peptide at the amino terminal side of the bacterial transglutaminase, and a DN encoding the fusion protein.
A plasmid having an A sequence, this DNA sequence and a promoter expressible in Escherichia coli, Escherichia coli harboring this plasmid, and culturing this E. coli to produce a bacterial transglutaminase fusion protein as an inactive inclusion body in the cells. , A bacterial transfection characterized by obtaining the fusion protein having transglutaminase activity by recovering the inclusion body from the bacterial cell, solubilizing the inclusion body with a denaturing agent, and then removing the denaturing agent. This is a method for producing glutaminase.

The present invention will be described in detail below. Generally, when proteins are produced in large quantities by microorganisms such as E. coli,
The proteins often associate to form inclusion bodies of the protein. This method of expression and production protects the target protein from digestion by protease inside the cell, or
It has the advantage that it can be easily purified by centrifugation after cell disruption.

The protein inclusion body thus obtained is solubilized by a protein denaturant, and after undergoing an activity regeneration operation mainly by removing the denaturant, it is transformed into a correctly folded physiologically active protein. Can be converted. For example, there are many examples such as activity regeneration of human interleukin-2 (Japanese Patent Laid-Open No. 61-257931).

[0016] In order to obtain the active protein from the protein inclusion body, a series of operations such as solubilization and active regeneration are necessary, which is more complicated than the direct production of the active protein. However, in the case of expressing and producing a protein that affects the growth of the bacterial cell, the effect can be suppressed by accumulating it in the bacterial cell as an inactive protein inclusion body.

As described above, as a method of mass-producing the target protein as an inclusion body, in addition to the method of expressing the target protein alone under the control of a strong promoter, it is possible to use a protein known to be expressed in large quantities. There is a method of expressing it as a fusion protein.

Further, it is also effective to dispose a recognition sequence for a restriction protease at an appropriate position in order to cut out a target protein after expressing it as a fusion protein. From this point of view, the method for producing BTG of the present invention comprises expressing a large amount of BTG as an inactive fusion protein inclusion body in Escherichia coli and efficiently regenerating it into an active body. The present invention will be described below.

<1> BTG Fusion Protein Expression System First, an expression system for producing BTG as an inactive fusion protein inclusion body in Escherichia coli will be described.

The BTG gene used in the present invention is not particularly limited, but it is B of actinomycete of the genus Streptoverticillium.
The TG gene may be mentioned. Plasmid pOMPA-BTG which has this gene and carries out secretory production to E. coli periplasm
Escherichia coli carrying (European Patent Publication No. 0481 504 A1: FIG. 1) has been deposited at the Institute of Microbial Technology, Institute of Industrial Technology (FERM BP-3558). The BTG gene can be recovered from this plasmid by cutting with Eco RI and Bam HI.

The BTG gene in the above plasmid is
Based on the amino acid sequence of BTG purified from Streptoverticillium spp., It was synthesized according to the codon usage of Escherichia coli and yeast and ligated downstream of the sequence encoding the ompA signal peptide of E. coli. Has been done.

As the promoter for expressing the above BTG gene, a promoter usually used for heterologous protein production in Escherichia coli can be used. For example, strong promoters such as T7 promoter, trp promoter, lac promoter, tac promoter and PL promoter can be used. A promoter is mentioned.

In order to produce BTG as a fusion protein inclusion body, another protein, preferably another gene encoding a hydrophilic peptide, is ligated upstream or downstream of the BTG gene to form a BTG fusion protein gene. To do. Such another gene may be any gene as long as it increases the accumulation amount of the fusion protein with BTG and enhances the solubility after the denaturation / regeneration process. For example, T7 gene 10 , β
-Galactosidase gene, dehydrofolate reductase gene, interferon γ gene, interleukin-2
A gene, a prochymosin gene, etc. are mentioned as a candidate.

The ligation of these genes with the BTG gene is carried out by ligation at an appropriate restriction enzyme site in which the reading frames of codons match, or synthetic DNA having an appropriate sequence.
Should be used.

In <1> in the examples described below, commercially available pGEMEX
A large amount of BTG fusion protein with T7 gene 10 was expressed as a protein inclusion body inactive in E. coli using a system vector (Promega). However, when the BTG fusion protein was solubilized with a denaturant and then the denaturant was removed by dialysis, the solubility was extremely decreased, and only a slight transglutaminase activity was observed in the soluble fraction.

Therefore, as described in <3> in the examples, when the highly hydrophobic sequence of the fusion protein portion was deleted, this BTG fusion protein was also expressed in large quantities as a protein inclusion body in E. coli. The fusion protein was highly soluble even after removal of the denaturant and was regenerated into an active form. Therefore, it is speculated that a hydrophilic protein is preferable for use in the fusion protein of the present invention,
Of the above-listed genes, those without the sequence corresponding to the hydrophobic portion may be used. The “hydrophilic peptide” in the present invention is a peptide excluding a highly hydrophobic sequence in this sense.

The vector for introducing the above fusion protein gene into Escherichia coli is preferably a so-called multicopy type, and examples thereof include a plasmid having a replication origin derived from Col E1, such as pUC type plasmid, pBR322 or its derivative. To be Moreover, in order to select transformants, it is preferable to have a marker such as an ampicillin resistance gene. As such a plasmid, an expression vector having a strong promoter is commercially available (pU
C series (Takara Shuzo and others), pPROK series (Clontech), pK
K233-2 (manufactured by Clontech) and others). It can be easily inferred that production is possible using any of these.

In order to increase the production amount, it is preferable to connect a terminator, which is a transcription termination sequence, downstream of the fusion protein gene. Examples of this terminator include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, Escherichia coli trpA gene terminator, and the like.

A plasmid in which a DNA fragment in which a promoter, a fusion protein gene, and a terminator are ligated in this order is inserted into the above vector is introduced into Escherichia coli, and this E. coli is cultured to express and produce the BTG fusion protein.

The nucleotide sequence encoding the BTG fusion protein of the present invention is exemplified in the sequence listing (SEQ ID NO: 1). A sequence in which the codon in this sequence is replaced with an equivalent codon can be used as well.

Further, even if a part of the sequence of the fusion protein portion derived from the sequences of the vectors shown in SEQ ID NOS: 1 and 2 of the Sequence Listing is deleted, inserted or substituted, it can be highly expressed as a protein inclusion body and the protein can be highly expressed. It can be regenerated by removing the denaturing agent. This is inferred from the examples described later.

Furthermore, it is considered possible to obtain a BTG fusion protein having transglutaminase activity by ligating the BTG gene to another high expression vector and using a method similar to the above. As a high expression vector, T7 gene
The expression vector plasmid that highly expresses the fusion protein of 10 and the linker peptide is Xpress from Invitrogen.
An expression vector plasmid under the trade name of System ^™ , which highly expresses a fusion protein with glutathione-S-transferase, is commercially available from Pharmacia LKB as a pGEX plasmid.

When expressed as a fusion protein, in addition to the blood coagulation factor Xa shown in the examples, kallikrein, etc.,
The transglutaminase may be excised by using a restriction protease having a recognition sequence that is a sequence that does not exist in BTG.

As Escherichia coli used in the expression system, a strain usually used for expressing a heterologous gene can be used, but Escherichia coli JM109 (DE3) strain and JM109 strain are particularly preferable.

<2> Expression production of BTG fusion protein by Escherichia coli The BTG fusion protein is produced by culturing Escherichia coli into which the above-mentioned BTG fusion protein-expressing plasmid has been introduced.

As the production medium, in addition to the M9-casamino acid medium described in the examples, a medium used for culturing ordinary Escherichia coli such as LB medium may be used. In addition, culture conditions, production induction conditions, the vector marker used, promoter,
It is appropriately selected depending on the type of host bacterium.

<3> Regeneration of activity from protein inclusion body The produced protein inclusion body is solubilized with a denaturing agent. Although it may be solubilized together with the bacterial protein, it is preferable to take out the inclusion body and solubilize it in consideration of purification and the like. The inclusion body may be recovered from the bacterial cells by a conventionally known method. For example, the microbial cells are destroyed and the inclusion bodies are collected by centrifugation or the like.

Examples of the denaturing agent for solubilizing the protein inclusion body include guanidine hydrochloric acid (eg, 6M, pH 5 to 8) and urea (eg, 8M). When these denaturants are removed by dialysis etc., they are regenerated as active proteins. As a dialysis solution used for dialysis, a Tris-hydrochloric acid buffer solution, a phosphate buffer solution, or the like may be used, and the concentration is 20 mM to 0.5 M, and the pH is 5 to 8.

The protein concentration during the regeneration step is 500 μg / ml
It is preferable to keep it below the level. The dialysis temperature is preferably 5 ° C. or lower in order to suppress self-crosslinking due to the regenerated BTG activity. Also, as a method of removing the denaturant,
In addition to this dialysis method, there are a dilution method, an ultrafiltration method, and the like, and the regeneration of activity can be expected by using either method.

[0040]

EXAMPLES Examples of the present invention will be described below together with comparative examples. The restriction enzymes used in this Example and Comparative Examples described later were products of Takara Shuzo Co., Ltd.

<1> Production of Fusion Protein of T7 gene 10 and BTG Escherichia coli ( E.
coli ) JM109 (DE3) -pGEMEX-1 (manufactured by Promega),
A fusion protein of the amino terminal portion of the T7 gene 10 product and BTG was expressed and produced.

In this system, a gene incorporated into the multicloning site downstream of the T7 promoter of the vector pGEMEX-1 is expressed in large quantities as a fusion protein with the T7 gene 10 product consisting of about 260 amino acid residues. The host for expression, E. coli JM109 (DE3), is expressed on the chromosome of E. coli JM109 under the control of the lacUV 5 promoter, T7.
It has an RNA polymerase gene (T7 gene 1 ) incorporated. Therefore, it is an inducer of the lacUV 5 promoter
Since T7 RNA polymerase is expressed in large quantities by the addition of IPTG (isopropyl-β-D-thiogalactopyranoside), T7
Expression of the gene inserted directly under the promoter is induced (Studier, F. and Moffatt, B. J. Mol . Biol ., 189 , 113
(1986)).

BTG fusion protein expression plasmid pGEM26
The construction method of 0BTG is shown in FIG. That is, the plasmid pOMPA-BTG which secretes BTG into the periplasm of E. coli.
(European Patent Publication No. 0 481 504 A1), the BTG gene cut out with the restriction enzymes Eco RI and Bam HI was added to pGEMEX.
-1 was inserted between Eco RI and Bam HI in the multiple cloning site. Escherichia coli JA2 carrying the plasmid pOMPA-BTG
Twenty-one strains have been deposited with the Institute of Microbial Technology, Institute of Industrial Technology (FERM BP-3558).

E. coli JM109 (DE3) transformed with pGEM260BTG thus constructed was shake-cultured at 37 ° C. in M9-casamino acid medium containing 50 μg / ml ampicillin, and OD
_{When 570} reaches 0.4, the final concentration will be 1 mM IP
TG was added, and the mixture was further cultured by shaking at 37 ° C for 3 hours.

After the whole cell extract was subjected to SDS-polyacrylamide gel electrophoresis, it was confirmed by Coomassie Brilliant Blue staining that a large amount of BTG fusion protein was expressed.

<2> Shortening of fusion protein part Next, in order to produce a BTG fusion protein that can be regenerated into an active form by being expressed in large amounts as an inactive protein inclusion body, In order to remove the T7 gene 10 part, a plasmid was prepared in which the sequence of the corresponding part in pGEM260BTG obtained in <1> was deleted.

The construction method is shown in FIG. First, pGEM260B
Cleavage TG with restriction enzyme Nhe I, and cut the cleaved end with DNA Blunting K
After smoothing with it (Takara Shuzo), a large fragment cut with Bam HI was recovered.

On the other hand, pGEM260BTG was cut with Nhe I, and the cut end was Exo III-Mungbean Deletion Kit (manufactured by Stratagene).
After shaving, the small fragment obtained by cutting with Bam HI was recovered. This small fragment and the above large fragment are ligated together, and E. col
i JM109 was transformed. Nhe among the obtained clones
A clone carrying a plasmid that was cut with I was selected. Since this Nhe I recognition site is located near the initiation codon, such a plasmid is convenient for the subsequent construction of a fused BTG expression plasmid.

Next, E. coli JM109 (DE3) transformed with these plasmids was cultured in the same manner as in <1>, and the bacterial cell extract was subjected to SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue. Staining and Western blotting with rabbit anti-BTG antibody were performed to obtain BTG.
A clone expressing a large amount of the fusion protein was selected.
The plasmid was recovered from the selected clones and the nucleotide sequence of the fusion protein gene was determined. This sequence is shown in SEQ ID NO: 2 in the Sequence Listing. In addition, the obtained plasmid was designated as pGEM.
77BTG.

This clone was inoculated into M9-casamino acid medium (casamino acid 0.2% (w / v)) containing 50 μg / ml ampicillin and precultured overnight at 37 ° C. This culture broth was inoculated to the same medium (however, casamino acid 2% (w / v)) at 5% and shake cultured at 37 ° C. When OD ₅₇₀ reached 0.4, the final concentration of IPTG was reached. It was added to 1 mM and further cultured for 3 hours.

The BTG fusion protein was subjected to SDS-polyacrylamide gel electrophoresis of the ultrasonically disrupted cell extract after culturing,
And, by Western blotting, a large amount was detected only in the insoluble fraction of bacterial protein. This is B
It shows that the TG fusion protein accumulated in the cells as a protein inclusion body.

From this insoluble fraction, a protein inclusion body was obtained by centrifugation at 10,000 × g for 10 minutes, and this was washed twice with 20 mM Tris-HCl (pH 7.5) -30 mM NaCl. This protein inclusion body was solubilized with 6 M guanidine hydrochloride-10 mM EDTA (pH 6.0), and then dialyzed against 200 mM Tris-hydrochloric acid (pH 6.0) at 5 ° C for one day.

The solubility of this BTG fusion protein in 200 mM Tris-HCl (pH 6.0) was extremely low, and solubilization could not be performed. Therefore, an assay for transglutaminase activity could not be performed.

<3> Production of fusion protein capable of regenerating activity In order to increase the solubility of the BTG fusion protein after denaturation-regeneration treatment, a sequence corresponding to the highly hydrophobic region of the T7 gene 10 product part in the sequence of pGEM77BTG was added. The deleted expression plasmid pGEM15BTG (Xa) was constructed. The construction method is shown in FIG. The nucleotide sequence and amino acid sequence of the portion encoding the fused BTG are shown in SEQ ID NO: 1 in Sequence Listing.

[0055] That is, cutting the pGEM77BTG with Nhe I and Pvu II, it was inserted synthetic DNA (corresponding to a nucleotide sequence of No. 5-73 in SEQ ID NO: 1). DNA synthesis was carried out by the phosphoamidite method using DNA Synthesizer 391 (Applied Biosystems).

The synthetic DNA sequence is present in the fusion protein portion in order to delete the above-mentioned highly hydrophobic portion and to avoid the formation of undesired disulfide bridges.
It was designed to replace Cys with Ser (amino acid number 4 in SEQ ID NO: 1). In addition, the recognition sequence Ile-Glu-Gly-Arg of Xa was added to B so that BTG can be excised from the fusion protein by the restriction protease blood coagulation factor Xa.
It was placed at the amino terminus of TG (amino acid numbers 12 to 15 in SEQ ID NO: 1). Further, Tyr in the vicinity of this Ile-Glu-Gly-Arg sequence was replaced with Gly (amino acid number 9 in SEQ ID NO: 1) so that the Ile-Glu-Gly-Arg sequence was exposed on the protein surface and was easily recognized and cleaved. Excision of the target protein from the fusion protein by cleavage with Xa has already been reported for β-globin and the like (K. Nagai, and HCThogersen, Nature , 309 , p810).
(1984)).

E. coli JM109 (DE3) (AJ12745) transformed with this plasmid pGEM15BTG (Xa) was deposited at the Institute of Microbial Technology, Institute of Industrial Technology (FERM P-13037). This transformant was inoculated into M9-casamino acid medium (0.2% (w / v) casamino acid) containing 50 μg / ml ampicillin, and left overnight at 37 ° C.
It was pre-cultured in. In the main culture, this was inoculated to the same medium (however, casamino acid 2% (w / v)) at 5% and shaken at 37 ° C. When OD ₅₇₀ reaches 0.4, IPTG final concentration is 1m
M was added to the mixture and the cells were further cultured for 3 hours.

The BTG fusion protein was subjected to SDS-polyacrylamide gel electrophoresis of the ultrasonically disrupted cell extract after culturing,
Also, it was confirmed by Western blotting that the protein was accumulated in the cells as inclusion bodies.

The collected cells were treated with 20 mM Tris-HCl (pH 7.5),
The cells were suspended in 30 mM NaCl and 50 mM EDTA and sonicated. From this, a protein inclusion body was obtained by centrifugation at 10,000 xg for 10 minutes, and this was added to 20 mM Tris-HCl (pH 7.5) -30 mM NaCl.
It was washed twice with and the protein inclusion bodies were collected.

6M guanidine hydrochloride-10 mM EDTA (pH 6.
After solubilization with 0), add 5 mM to 200 mM Tris-HCl (pH 6.0).
Dialysis was performed by a batch method and a dropping dialysis method at 24 ° C. overnight.

The solubility of this fusion protein after removal of the denaturant was about 400 μg / ml. The activity of the regenerated BTG was measured by a method for measuring transglutaminase activity using hydroxamic acid (colorimetric hydroxamate procedure: JEFolka).
nd PWCole, J. Biol . Chem ., 241 , 5518 (1966), modified).

Using benzyloxycarbonyl-L-glutamylglycine and hydroxylamine as substrates, Ca
^The reaction was performed in the presence or absence of ⁺² , the formed hydroxamic acid was formed as an iron complex in the presence of trichloroacetic acid, the absorption at 525 nm was measured, and the amount of hydroxamic acid was determined from the calibration curve to calculate the activity. However, the activity of regenerated BTG was 3.2 Units / mg protein. The activity of the periplasmic fraction was 0.22 when BTG was secreted and produced.
U / mg protein (European Patent Publication 0 481 50
4 A1), the present invention enabled to obtain 10 times or more of activity.

Further, BTG was cut out with the blood coagulation factor Xa. Add 1 μg of blood coagulation factor Xa (Danex Biotech) to 10 μg of fusion protein after dialysis, and
00 mM Tris-HCl solution (pH 6) (100 mM NaCl, 1 mM CaCl ₂ )
The reaction was carried out at 5 ° C overnight.

After the blood coagulation factor Xa reaction, it was confirmed by SDS-polyacrylamide gel electrophoresis and Western blotting that BTG was excised. Transglutaminase activity did not change before and after cleavage, but T7 g
Since you can remove unnecessary parts derived from ene 10 ,
It is particularly useful in the food field and the like.

[0065]

[Comparative example]

(Production of BTG Using trc Promoter) As a comparative example, an example of producing BTG by controlling the trc promoter which is one of strong promoters will be described.

POMPA-BTG (European Patent Publication 0 48
BT excised with restriction enzymes Eco RI and Bam HI from 1 504 A1)
The G gene was inserted between Eco RI and Bam HI in the multiple cloning site downstream of the trc promoter of the commercially available expression plasmid pTrc99A (Pharmacia).

E. coli JM109 transformed with pTrc-BTG thus constructed contains 50 μg / ml of ampicillin.
Cultured in M9-casamino acid medium at 37 ° C with shaking to obtain OD ₅₇₀ of 0.4
The culture temperature was lowered to 23 ° C. And tr
IPTG, which is an inducer of the c promoter, was added so that the final concentration was 1 mM, and the mixture was further shake-cultured for 3 hours. The M9
-The casamino acid medium is M9 medium (JH Miller ed., Experimen
ts in Molecular Genetics (Cold Spring Harbor, 197
2)) was added with casamino acid (VITAMIN ASSAY CASAMINO ACID (manufactured by Difco)) to a concentration of 2% (w / v).

The microbial cell extract after culturing was subjected to SDS-polyacrylamide gel electrophoresis, and then the gel was stained with Coomassie Brilliant Blue, but this gene product was not detected. However, this gene product was confirmed by Western blotting using a rabbit anti-BTG antibody, and the production amount was about 20 mg / l (medium) when estimated from the transglutaminase activity measured using hydroxamic acid.

[0069]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1044 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA and vector-derived D
NA sequence ATG GCT AGC TCT GAC CCA GAT AGC GGC TCT GCG ATT GAA GGT CGC GAT 48 Met Ala Ser Ser Asp Pro Asp Ser Gly Ser Ala Ile Glu Gly Arg Asp 1 5 10 15 TCT GAC GAT CGA GTC ACT CCA CCA GCT GAA CCA TTG GAT AGA ATG CCA 96 Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro 20 25 30 GAT CCA TAC AGA CCA TCT TAC GGT AGA GCT GAA ACT GTT GTC AAC AAC 144 Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn Asn 35 40 45 TAC ATT AGA AAG TGG CAA CAA GTC TAC TCT CAC AGA GAT GGT AGA AAG 192 Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys 50 55 60 CAA CAA ATG ACT GAA GAA CAA AGA GAA TGG TTG TCT TAC GGT TGT GTT 240 Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val 65 70 75 80 GGT GTT ACT TGG GTT AAC TCT GGT CAA TAC CCA ACT AAC AGA TTG GCT 288 Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala 85 90 95 TTC GCT TCT TTC GAT GAA GAT AGA TTC AAG AAC GAA TTG AAG AAC GGT 336 Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly 100 105 11 0 AGA CCA AGA TCC GGT GAA ACT AGA GCT GAA TTC GAA GGT AGA GTT GCT 384 Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala 115 120 125 AAG GAA TCT TTC GAT GAA GAA AAG GGT TTC CAA AGA GCT AGA GAA GTT 432 Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val 130 135 140 GCT TCT GTT ATG AAC AGA GCT CTA GAA AAC GCT CAC GAT GAA TCT GCT 480 Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala 145 150 155 160 TAC TTG GAT AAC TTG AAG AAG GAA TTG GCC AAC GGT AAC GAT GCT TTG 528 Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu 165 170 175 AGA AAC GAA GAT GCT AGA TCC CCA TTC TAC TCT GCT TTG AGA AAC ACT 576 Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr 180 185 190 CCA TCT TTC AAG GAA AGA AAC GGT GGT AAC CAC GAT CCA TCC AGA ATG 624 Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met 195 200 205 AAG GCT GTT ATT TAC TCT AAG CAC TTC TGG TCT GGT CAA GAT AGA TCT 672 Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Se r 210 215 220 TCT TCT GCT GAT AAG AGA AAG TAC GGT GAT CCA GAT GCT TTC AGA CCA 720 Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Pro 225 230 235 240 GCT CCA GGT ACC GGT TTG GTC GAC ATG TCC AGA GAT AGA AAC ATT CCA 768 Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro 245 250 255 AGA TCC CCA ACT TCT CCA GGT GAA GGT TTC GTC AAC TTC GAT TAC GGT 816 Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly 260 265 270 TGG TTC GGT GCT CAA ACT GAA GCT GAT GCT GAT AAG ACT GTT TGG ACC 864 Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr 275 280 285 CAT GGT AAC CAC TAC CAC GCT CCA AAC GGT TCT TTG GGT GCT ATG CAC 912 His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His 290 295 300 GTC TAC GAA TCT AAG TTC AGA AAC TGG TCT GAA GGT TAC TCT GAT TTC 960 Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp Phe 305 310 315 320 GAT AGA GGT GCT TAC GTT ATT ACT TTC ATT CCA AAG TCT TGG AAC ACT 1008 Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro L ys Ser Trp Asn Thr 325 330 335 GCT CCA GAC AAG GTC AAG CAA GGT TGG CCA TAATGA 1044 Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 340 345

SEQ ID NO: 2 Sequence length: 1230 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA and vector-derived D
NA sequence ATG GCT AGC TGT GAC CCA GAT AGC TAC TCT GCG ATT CTG GCA GCA CTG 48 Met Ala Ser Cys Asp Pro Asp Ser Tyr Ser Ala Ile Leu Ala Ala Leu 1 5 10 15 ATG CCG AAC GCA GCA AAC TAC GCT GCT CTG ATT GAC CCT GAG AAG GGT 96 Met Pro Asn Ala Ala Asn Tyr Ala Ala Leu Ile Asp Pro Glu Lys Gly 20 25 30 TCT ATC CGC AAC GTT ATG GGC TTT GAG GTT GTA GAA GTT CCG CAC CTC 144 Ser Ile Arg Asn Val Met Gly Phe Glu Val Val Glu Val Pro His Leu 35 40 45 ACC GCT GGT GGT GCT GGT ACC GGA TCG AAT TGG CCA AGT TTA TTA ACC 192 Thr Ala Gly Gly Ala Gly Thr Gly Ser Asn Trp Pro Ser Leu Leu Thr 50 55 60 CTC ACT AAA GGG AAG GCC AAG TCG GCC GAG CTC GAA TTC GAT TCT GAT 240 Leu Thr Lys Gly Lys Ala Lys Ser Ala Glu Leu Glu Phe Asp Ser Asp 65 70 75 80 GAC AGA GTC ACT CCA CCA GCT GAA CCA TTG GAT AGA ATG CCA GAT CCA 288 Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro 85 90 95 TAC AGA CCA TCT TAC GGT AGA GCT GAA ACT GTT GTC AAC AAC TAC ATT 336 Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile 100 105 11 0 AGA AAG TGG CAA CAA GTC TAC TCT CAC AGA GAT GGT AGA AAG CAA CAA 384 Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln 115 120 125 ATG ACT GAA GAA CAA AGA GAA TGG TTG TCT TAC GGT TGT GTT GGT GTT 432 Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val 130 135 140 ACT TGG GTT AAC TCT GGT CAA TAC CCA ACT AAC AGA TTG GCT TTC GCT 480 Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala 145 150 155 160 TCT TTC GAT GAA GAT AGA TTC AAG AAC GAA TTG AAG AAC GGT AGA CCA 528 Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro 165 170 175 AGA TCC GGT GAA ACT AGA GCT GAA TTC GAA GGT AGA GTT GCT AAG GAA 576 Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu 180 185 190 TCT TTC GAT GAA GAA AAG GGT TTC CAA AGA GCT AGA GAA GTT GCT TCT 624 Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser 195 200 205 GTT ATG AAC AGA GCT CTA GAA AAC GCT CAC GAT GAA TCT GCT TAC TTG 672 Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Le u 210 215 220 GAT AAC TTG AAG AAG GAA TTG GCC AAC GGT AAC GAT GCT TTG AGA AAC 720 Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn 225 230 235 240 GAA GAT GCT AGA TCC CCA TTC TAC TCT GCT TTG AGA AAC ACT CCA TCT 768 Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser 245 250 255 TTC AAG GAA AGA AAC GGT GGT AAC CAC GAT CCA TCC AGA ATG AAG GCT 816 Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala 260 265 270 GTT ATT TAC TCT AAG CAC TTC TGG TCT GGT CAA GAT AGA TCT TCT TCT 864 Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser 275 280 285 GCT GAT AAG AGA AAG TAC GGT GAT CCA GAT GCT TTC AGA CCA GCT CCA 912 Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Pro Ala Pro 290 295 300 GGT ACC GGT TTG GTC GAC ATG TCC AGA GAT AGA AAC ATT CCA AGA TCC 960 Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser 305 310 315 CCA ACT TCT CCA GGT GAA GGT TTC GTC AAC TTC GAT TAC GGT TGG TTC 1008 Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp T yr Gly Trp Phe 320 325 330 GGT GCT CAA ACT GAA GCT GAT GCT GAT AAG ACT GTT TGG ACC CAT GGT 1056 Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly 335 340 345 AAC CAC TAC CAC GCT CCA AAC GGT TCT TTG GGT GCT ATG CAC GTC TAC 1104 Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr 350 355 360 GAA TCT AAG TTC AGA AAC TGG TCT GAA GGT TAC TCT GAT TTC GAT AGA 1152 Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp Phe Asp Arg 365 370 375 GGT GCT TAC GTT ATT ACT TTC ATT CCA AAG TCT TGG AAC ACT GCT CCA 1200 Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro 380 385 390 395 GAC AAG GTC AAG CAA GGT TGG CCA TAATGA 1230 Asp Lys Val Lys Gln Gly Trp Pro 400

[0072]

INDUSTRIAL APPLICABILITY According to the present invention, by producing BTG as an inactive fusion protein inclusion body, it became possible to mass-produce BTG which was not highly expressed in E. coli conventionally. Moreover, it became possible to obtain active BTG from this inactive fusion protein inclusion body. Furthermore, the present invention opens the way to genetic engineering / protein engineering analysis of BTG, and thus enables mass production of improved BTG.

[Brief description of drawings]

FIG. 1 is a construction diagram of an expression plasmid pGEM260BTG.

FIG. 2 is a construction diagram of an expression plasmid pGEM77BTG.

FIG. 3 is a construction diagram of the expression plasmid pGEM15BTG (Xa).

Claims (8)

[Claims] 1. One of the amino acid sequences shown in SEQ ID NO: 1
A fusion protein comprising a bacterial transglutaminase having an amino acid sequence of 6 to 346 and a hydrophilic peptide on the amino terminal side of the bacterial transglutaminase. 2. The fusion protein according to claim 1, which has the amino acid sequence shown in SEQ ID NO: 1. 3. One of the amino acid sequences shown in SEQ ID NO: 1
A DNA sequence encoding a fusion protein comprising a bacterial transglutaminase having an amino acid sequence of 6 to 346 and a hydrophilic peptide on the amino terminal side of the bacterial transglutaminase. 4. The DNA sequence according to claim 3, wherein a D coding for a recognition sequence for a restriction protease is located between the sequence part encoding the bacterial transglutaminase and the sequence part encoding the hydrophilic peptide.
A DNA sequence having an NA sequence. 5. The DNA sequence according to claim 3, which has the nucleotide sequence shown in SEQ ID NO: 1. 6. D according to any one of claims 3 to 5.
A plasmid having an NA sequence and a promoter expressible in E. coli. 7. Escherichia coli carrying the plasmid according to claim 6. 8. A bacterial transglutaminase fusion protein is produced as an inactive inclusion body in the cells by culturing the Escherichia coli according to claim 7,
Bacterial transglutaminase characterized in that a fusion protein having transglutaminase activity is obtained by recovering the inclusion body from the bacterial cell, solubilizing the inclusion body with a denaturing agent, and then removing the denaturing agent. Manufacturing method.