JP3010589B2 - Recombinant transglutaminase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DNA gene encoding transglutaminase, a plasmid incorporating the gene, a transformant into which the plasmid has been introduced, and culturing the transformant. The present invention relates to a method for producing transglutaminase.

[0002]

2. Description of the Related Art Transglutaminase (hereinafter referred to as "BT")
G ”. ) Is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine residue in a peptide chain.

This transglutaminase, when the ε-amino group of a lysine residue in a protein acts as an acyl receptor, ε- (γ-Gln) -Lys within and between molecules.
Crosslinks are formed. When water functions as an acyl acceptor, the enzyme promotes a reaction in which a glutamine residue is deamidated into a glutamic acid residue.

[0004] Transglutaminase is used as a gel food,
It is used to manufacture gel cosmetics, yogurt, jelly, cheese, etc.
No. 82).

[0005] Further, it is also used for producing heat-stable materials for microcapsules, carriers for immobilized enzymes and the like.

[0006] Transglutaminase has been known to be derived from animals. For example, guinea pig liver [Co
nnellan, et al., Journal of Biological Chemistry
246: 4, 1093-1098 (1971)] and widely distributed in mammalian organs and blood [Folk et al., Advances in Enz.
ymology 38, 109-191 (1973); Folk et al., A
dvances in Protein Chemystry 31, 31-133 (197
7)], and the characteristics of the enzyme have been studied.

Further, a calcium (Ca ²⁺ ) -independent transglutaminase has been discovered from bacteria of the genus Streptoverticillium, unlike transglutaminase derived from the above animals. Specific examples of the genus bacteria include Streptoverticillium glyceocarneum (Strepto
verticillium griseocarneum) IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum subsp. c
innamoneum) IFO 12852, Streptoverticillium mobaraense IFO 13
819 and the like (see JP-A-64-27471).

[0008]

Conventionally, since transglutaminase has been produced from animals, fungi and the like, there have been many points to be improved in terms of supply amount, supply cost and the like.

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the DN encoding transglutaminase
A was purified and isolated, and its base sequence was successfully determined. Based on such a result, the DNA is expressed using a microorganism such as Escherichia coli by a genetic engineering technique, thereby opening the way to efficient mass production of transglutaminase.

[0010]

Accordingly, the present invention provides a DNA encoding transglutaminase.

That is, the present invention provides a DNA comprising a base sequence encoding the amino acid sequence shown in Table 1 below when represented by the one-letter symbol of the amino acid.

[0012]

[Table 6] Such DNA can include various nucleotide sequences in consideration of the degeneracy of genetic codons.

These nucleotide sequences can be easily selected by those skilled in the art based on various elements of the gene expression system, for example, preferential codons depending on the type of host cell and the like.

As a specific example, an example of a base sequence suitable for an expression system using Escherichia coli or yeast as a host cell is shown in Table 2 (SEQ ID NO: 1).

[0015]

[Table 7] Table 2 GAT TCT GAT GAC AGA GTC ACT CCA CCA GCT GAA CCA TTG GAT AGA ATG CCA GAT CCA TAC AGA CCA TCT TAC GGT AGA GCT GAA ACT GTT GTC AAC AAC TAC ATT AGA AAG TGG CAA CAA GTC TAC TCT CAC AGA GAT GGT AGA AAG CAA CAA ATG ACT GAA GAA CAA AGA GAA TGG TTG TCT TAC GGT TGT GTT GGT GTT ACT TGG GTT AAC TCT GGT CAA TAC CCA ACT AAC AGA TTG GCT TTC GCT TCT TTC GAT GAA GAT AGA TTC AAG TTG AAG AAC GGT AGA CCA AGA TCC GGT GAA ACT AGA GCT GAA TTC GAA GGT AGA GTT GCT AAG GAA TCT TTC GAT GAA GAA AAG GGT TTC CAA AGA GCT AGA GAA GTT GCT TCT GTT ATG AAC AGA GCT CTA GAA AGA GCT CAC GAT TCT GCT TAC TTG GAT AAC TTG AAG AAG GAA TTG GCC AAC GGT AAC GAT GCT TTG AGA AAC GAA GAT GCT AGA TCC CCA TTC TAC TCT GCT TTG AGA AAC ACT CCA TCT TTC AAG GAA AGA AAC GGT GGT AAC CAC GAT CCA TGA AGA AAG GCT GTT ATT TAC TCT AAG CAC TTC TGG TCT GGT CAA GAT AGA TCT TCT TCT GCT GAT AAG AGA AAG TAC GGT GAT CCA GAT GCT TTC AGA CCA GCT CCA GGT ACC GGT TTG GTC GAC ATG TCC AGA GAT AGA AAC ATT CCA AGA CCA ACT TCT CCA GGT GAA GGT TTC GTC AAC TTC GAT TAC GGT TGG TTC GGT GCT CAA ACT GAA GCT GAT GCT GAT AAG ACT GTT TGG ACC CAT GGT AAC CAC TAC CAC GCT CCA AAC GGT TCT TTG GGT GCT ATG CAC GTC TAC GACT TTC AGA AAC TGG TCT GAA GGT TAC TCT GAT TTC GAT AGA GGT GCT TAC GTT ATT ACT TTC ATT CCA AAG TCT TGG AAC ACT GCT CCA GAC AAG GTC AAG CAA GGT TGG CCA It can be prepared by a synthetic method or the like.

The present invention further provides a base sequence encoding all or a part of the amino acid sequence containing the signal peptide shown in Table 3 below at the 5 'end of the DNA encoding the amino acid sequence shown in Table 1. And DNA containing the same.

[0017]

[Table 8] The above DNAs can also include various base sequences due to the degeneracy of gene codons. As a specific example, those having the following base sequence (SEQ ID NO: 2) at the 5 'end of the base sequence shown in Table 2 can be mentioned.

[0018]

[Table 9] AAGAGAAGATCTCCAACTCCAAAGCCAACTGCTTCTAGAAGAATGACTTCTAGACACCAA AGAGCTCAAAGATCTGCTCCAGCTGCTTCTTCTGCTGGTCCATCTTTCAGAGCTCCA Also, various methods for obtaining the same including a chemical synthesis method can be considered. For example, PCR (Polymerase Chain Rea
ction) using a DNA fragment obtained as a probe and cloning from genomic DNA or the like of actinomycetes. Table 4 shows an example of the thus obtained transglutaminase structural gene and the nucleotide sequence of the DNA containing the 5 'terminal upstream portion.

[0019]

[Table 10] Table 4 ATGCGCTATA CGCCGGAGGC TCTCGTCTTC GCCACTATGA GTGCGGTTTA TGCACCGCCG GATTCATGCC GTCGGCCGGC GAGGCCGCCG CCGACAATGG CGCGGGGGAA GAGACGAAGT CCTACGCCGA AACCTACCGC CTCACGGCGG ATGACGTCGC GACATCAACG CGCTCAACGA AGCGCTCCGG CCGCTTCGAG CGCCGGCCCG TCGTTCCGGG CCCCCGACTC CGACGACAGG GTCACCCCTC CCGCCGAGCC GCTCGACAGG ATGCCCGACC CGTACCGTCC CTCGTACGGC AGGGCCGAGA CGGTCGTCAA CAACTACATA CGCAAGTGGC AGCAGGTCTA CAGCCACCGC GACGGCAGGA AGCAGCAGAT GACCGAGGAG CAACGGGAGT GGCTGTCCTA CGGCTGCGTC GGTGTCACCT GGGTCAATTC GGGTCAGTAC CCCACGAACA GACTGGCCTT CGCGTCCTTC GACGAGGACA GGTTCAAGAA CGAGCTGAAG AACGGCAGGC CCCGGTCCGG CGAGACGCGG GCGGAGTTCG AGGGCCGCGT CGCGAAGGAG AGCTTTGATG AAGAGAAGGG GTTCCAGCGG GCGCGTGAGG TGGCGTCCGT GATGAACAGG GCCCTGGAGA ACGCCCACGA CGAGAGCGCT TACCTCGACA ACCTCAAGAA GGAACTGGCG AACGGCAACG ACGCCCTGCG CAACGAGGAC GCCCGTTCCC CGTTCTACTC GGCGCTGCGG AACACGCCGT CCTTTAAGGA GCGGAACGGA GGCAATCACG ACCCGTCCAG GATGAAGGCC GTCATCTACT CGAAGCACTT CTGGAGCGGC CAGGACCGGT CGAGTTCGGC CGACAAGAGG AAGTACGGCG ACCC GGACGC TTTCCGCCCG GCCCCCGGGA CCGGCCTGGT CGACATGTCG AGGGACAGGA ACATTCCGCG CAGCCCCACC AGCCCCGGTG AGGGATTCGT CAATTTCGAC TACGGCTGGT TCGGCGCCCA GACGGAAGCG GACGCCGACA AGACCGTCTG GACCCACGGA AATCACTATC ACGCGCCCAA TGGCAGCCTT GGTGCCATGC ATGTATACGA GAGCAAGTTC CGCAACTGGT CCGAAGGTTA CTCCGACTTC GACCGCGGAG CCTATGTGAT CACCTTCATC CCCAAGAGCT GGAACACCGC CCCCGACAAG GTAAAGCAGG GCTGGCCG present invention also relates to the use for the expression and secretion of transglutaminase It provides a vector that can be used.

Such a vector can be prepared by inserting a DNA containing the nucleotide sequence shown in Tables 1 to 4 into a known expression vector according to a desired expression system by a conventionally known method. It is.

Examples of the known expression vectors which can be used for the preparation of the expression secretion vector of the present invention, mention may be made of PIN-III -ompA ₂ such as, for example, for E. coli hosts. As a specific example of the expression secretion vector of the present invention obtained by incorporating the DNA of the present invention to PIN-III -ompA ₂ is
There is pOMPA-BTG. Furthermore, for actinomycete hosts, pIJ7
02, pNJ1053 is used for yeast hosts. Specific examples of the expression secretion vector of the present invention obtained by incorporating the DNA of the present invention into these expression vectors include pIJ702-BT
G, pNJ1053-proBTG and pNJ1053-BTG.

Further, the present invention relates to various transformed transformants obtained by introducing an expression secretion vector carrying a transglutaminase gene.

The cells which can be the transformants include prokaryotic cells such as Escherichia coli and actinomycetes and eukaryotic cells such as yeast.

A specific example of E. coli is strain JA221 (hs
dM ⁺ , trpE5, leuB6, lacY, recA / F ′, lacI ^q , lac ⁺ ,
pro ⁺ ).

The transformant AJ12569 obtained by introducing the vector pOMPA-BTG of the present invention into the E. coli strain JA221 was transformed into AJ12569 on September 28, 1990 by the Research Institute of Microbial Industry and Technology of the Agency of Industrial Science and Technology. (Accession number FERM BP-3558).

One specific example of actinomycetes is Streptomyces
There is Streptomyces lividans 3131-TS obtained as a thiostrepton-sensitive strain from lividans 3131.

The transformant Streptomyces livid obtained by introducing the vector pIJ702-BTG of the present invention into such Streptomyces lividans 3131-TS is transformed.
ansAKW-1 has been deposited on September 30, 1991 with the Research Institute for Microbial Industry and Technology (Accession No. FERM BP-
3586).

One specific example of yeast is Saccharomyces c
erevisiae KSC22-1C (MATa, ssl1, leu2 , his -, ura
3) There is.

Such Saccharomyces cerevisiae KSC 22
Transformant Saccharo obtained by introducing the vector pNJ1053-proBTG of the present invention into -1C
myces cerevisiae AJ 14669 has been deposited on September 30, 1991 with the Research Institute of Microbial Industry and Technology (Accession No. FERM BP-3585).

Finally, the present invention relates to a method for producing a protein having a transglutaminase activity by culturing the above transformant.

Culture conditions can be appropriately determined by those skilled in the art according to the type of the transformant. In addition, I
By adding a substance such as PTG to the medium, the expression of the desired gene can be induced. The expressed and secreted protein can be isolated and purified from the culture supernatant and the periplasm of the host cell, and further from the cytoplasm by various conventionally known methods.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0033]

EXAMPLES Example 1: Cloning of BTG gene (1) Acquisition of bacterial cells Streptoverticillium sp.
C. for 5 days.

[GP medium] Glycerol 0.4 wt% Peptone 0.1 wt% Yeast extract 0.4 wt% Magnesium sulfate 0.05 wt% Monopotassium phosphate 0.2 wt% Disodium phosphate 0.5 wt% Glycine 0.1 wt% / 1l (2) Bacteria Obtaining DNA from the body After culturing under the above conditions, 400 ml of the culture medium is centrifuged (12,000 g, 4 ° C., 10 minutes), and this is centrifuged at 50 mM Tris-HCl
(pH 8.0) -5 mM EDTA-50 mM NaCl (hereinafter referred to as “TES”). The suspension is then centrifuged (1,10
(0 g, room temperature, 10 minutes), and the supernatant was discarded. The resulting precipitate (cells) was washed with 2 mg / ml lysozyme (Sigma) in T
The suspension was suspended in 5 ml of ES and incubated at 37 ° C. for 1 hour. After the incubation, this was placed in acetone-dry ice and rapidly frozen, and 42 ml of 100 mM Tris-HCl (pH 9.0) was added.
-1% SDS-100 mM NaCl (hereinafter referred to as "Tris-SDS") was added thereto, and the mixture was well suspended. This was further incubated at 60 ° C. for 20 minutes and immersed in acetone-dry ice for 10 minutes.
Next, after incubating it again at 60 ° C,
Extracted with phenol saturated with Tris-SDS. This extraction was repeated twice, and twice the volume of ethanol was added to the obtained product. At this time, the DNA of the cells becomes filamentous,
This was wound up and collected with a glass rod. The recovered DNA
It was washed once with 80% ethanol and dried using an aspirator and a desiccator. After drying, the DNA was washed with 10 mM Tris-HC.
l (pH8.0) -1mM EDTA (hereinafter referred to as "TE").

Next, the RNA in the DNA sample was decomposed and removed. For RNA degradation, RNase A (Sigma) 1 mg / ml and RNase TI were added to a sample in which DNA was dissolved in 5 ml of TE.
(Boehringer Mannheim) A solution containing 2000 u / ml
This was performed by adding 0.5 ml and incubating at 37 ° C. for 30 minutes. After the incubation, the sample was extracted once with TE-saturated phenol / chloroform and chloroform, and 1/10 volume of a 3 M sodium acetate (pH 5.2) solution and 2 volumes of ethanol were added to the finally obtained aqueous layer. −80
Placed at 30 ° C for 30 minutes. Then, centrifuge (12,000 g, 4
(C, 15 minutes), and the precipitate was washed with 70% ethanol and dried. This was dissolved in 4 ml of TE and used for the following experiments. The final amount of DNA is about 4mg
Met.

(3) PCR (Polymerase Chain Reactio)
n) Obtaining DNA fragment by the method A specific DNA region containing the BTG gene was isolated and amplified by a recently developed PCR method (Saiki, RFet).
al. (1985) Science 230, 1350-1354, Mullis, KB and Faloona, FA (1987) Methods in Enzymology, 155,
335-350).

(I) Synthesis of Primer DNA used for PCR method Base sequence corresponding to phenylalanine at position 117 to phenylalanine at position 123 of the amino acid sequence of BTG protein, which was determined by Shimonishi Laboratory, Institute for Protein Research, Osaka University. And DNA was synthesized. (The DNA was synthesized using a cyclone plus DNA synthesizer manufactured by Milligen Biosearch.) This DNA was used as Primer # 1 for PCR.

The sequence of Primer # 1 is shown below.

[0039]

[Table 11]

Next, a nucleotide sequence corresponding to lysine at position 325 to proline at position 331 was predicted, and DNA was synthesized in the same manner. This DNA was used as PCR Primer # 2. Pri
The sequence of mer # 2 is shown below.

[0041]

[Table 12]

Each of the synthesized DNAs was dissolved in TE to a concentration of 20 μM.

(Ii) The amplification reaction of the DNA fragment by the PCR method was performed using Gene Amp of PerkinElmer Japan, Inc.
Using the ^TM kit, the company's DNA Thermal Cycler (DNA
Amplifying device). The composition of the reaction solution is as follows.

[0044]

(Final concentration) 53.5 μl of H ₂ O [10x] Reaction buffer 10 μl [1x] dNTPs, Mix 1.25 mM 16 μl 200 μM (i) Primer # 1 5 μl 1.0 μM (i) Primer # 2 5 μl 1.0 μM * template (BTG DNA 0.5 μg) 10 μl AmpliTaq ^™ DNA polymerase 0.5 μl 2.5 u / assay total 100 μl * The DNA obtained in (2) was dissolved in TE to 0.5 μg / 10 μl.

100 μl of the above reaction mixture was mixed, and 100 μl of mineral oil (Sigma) was added. Next, the tube containing the reaction solution was set in a DNA thermal cycler, and the reaction was performed under the following conditions.

The reaction was performed 35 cycles under these conditions at 95 ° C. for 1 minute, at 37 ° C. for 2 minutes, at 72 ° C. for 3 minutes, and further incubated at 72 ° C. for 7 minutes.

(Iii) Recovery of amplified DNA After the reaction, the mineral oil was removed, 100 μl of chloroform was added and mixed, and the mixture was centrifuged at 15,000 rpm for 2 minutes (manufactured by Tommy Seiko Co., Ltd.). Was collected in an amount of 100 μl. Using 10 μl of these, the size and amount of DNA recovered by 1.5% agarose electrophoresis were confirmed. As a result, it was found that about 2 μg of the 645 bp DNA fragment was amplified.

The remaining 90 μl was subjected to 1.5% low melting point agarose electrophoresis, and a band corresponding to 645 bp was cut out.
After melting at ℃, an equal amount of phenol was added and mixed. After centrifugation, the aqueous layer was further treated with phenol / chloroform and chloroform sequentially, 3M sodium acetate was added to the aqueous layer to 8%, and ethanol was added. Double the amount,
Placed at -80 ° C for 15 minutes. Next, 15,000 rpm for 10 minutes 4
After centrifugation at ℃, the precipitate was dissolved in 20 μl of water. By this operation, about 1 μg of the DNA fragment was recovered.

(4) Confirmation of Structure of DNA Amplified by PCR In order to confirm whether the DNA fragment amplified by the above PCR is part of the BTG gene,
A direct sequence was performed using 0.4 μg. The method is shown below, and the above-mentioned # 1 used for PCR was used as a primer for the sequence.

-Preparation- 1. DNA sequencing reagent: basically dideoxy
Method was used. Sequenase ^™ (version 2.0), a sequencing kit from USB, USA, was used.

2. Labeling of primers for sequencing: 2 pmol of primer # 1 used for the PCR reaction was prepared, and the 5 'end was labeled with [ ³² P] with T4 Kinase (TOYOBO) (specific activity 3000 Ci / mmol [γ- ³² P] ATP). Free [γ- ³² P] ATP was removed from the labeled primer through an appropriate mini column or the like.

3. Sequence reaction solution (in 3.25 μl): S
G, A, each in 4 tubes using equenase Kit
Those for the T and C reactions were separately prepared.

2.5 μl G, A, T, C, termination mix 0.38 μl 5 × buffer 0.22 μl 0.1 M DTT 0.15 μl Sequenase (2 units) -Reaction- 1. 0.4 μg of amplified DNA and 2 pmol of [ ³² P The labeled primer was dissolved in 12 μl of TE, heat-denatured at 95 ° C. for 5 minutes, and quenched in ice water.

2. Immediately, 2.8 μl of the solution of (1) was placed in four tubes, and 3.25 μl of each sequencing reaction solution was added, followed by incubation at 37 ° C. for 10 minutes. After adding 4 μl of the reaction stop solution and heating at 75 to 80 ° C. for 2 minutes, the mixture was electrophoresed on a sequence gel.

As a result of the direct sequence, a nucleotide sequence encoding the amino acid sequence from the 129th valine to the 149th asparagine in the amino acid sequence of BTG was found in this fragment. Therefore, this fragment was assumed to be a part of the BTG gene.

(5) pU of DNA fragment amplified by PCR
Subcloning into C19 Next, the DNA fragment amplified by PCR was subcloned into the SmaI site of pUC19. For this purpose, both ends of the DNA fragment obtained by PCR were first blunted. The smoothing reaction is
It carried out as follows using Blunting kit (Takara Shuzo).

1. The following reaction solution was prepared in a microcentrifuge tube, and the total volume was adjusted to 9 μl.

8 μl of DNA fragment (0.4 μg) 1 μl of 10 × buffer 2. In order to prevent annealing of DNA ends, the mixture was kept at 70 ° C. for 5 minutes and then transferred to a thermostat at 37 ° C.

3. 1 μl of T4 DNA polymerase was added and gently mixed by pipetting.

4. Incubated at 37 ° C. for 5 minutes.

5. Add DNA dilution buffer to a final concentration of 1μ.
g DNA / 50 μl and vortexed vigorously.

The obtained DNA fragment was ligated with pUC19 digested with SmaI and ligated into Escherichia coli DH5α.
With 5-bromo-1-chloro-3-indolyl-β-D
-Galactoside (X-gal) and isopropyl-β-D-
Transformation was performed in the presence of thiogalactoside (IPTG). A plasmid obtained by integrating a DNA fragment amplified by PCR into an SmaI site and obtained from an ampicillin-resistant white colony was named pUC 19 BTG and used in the following experiments. In addition, ligation kit (Takara Shuzo)
Was used.

Next, in order to know a wider range of base sequence of the DNA fragment amplified by PCR, a DNA sequence was performed using the above-mentioned pUC19BTG as a template. DNA sequencing is a 7-deaza dGTP specification sequence kit (USB
The method was performed by a conventionally known method using the method described in US Pat. As a result, the base sequence of 564 bp shown in Table 5 below was revealed.

[0064]

[Table 14] Table 5 TCCGTGATGA ACAGGGCCCT GGAGAACGCC CACGACGAGA GCGCTTACCT CGACAACCTC AAGAAGGAAC TGGCGAACGG CAACGACGCC CTGCGCAACG AGGACGCCCG TTCCCCGTTC TACTCGGCGC TGCGGAACAC GCCGTCCTTT AAGGAGCGGA ACGGAGGCAA TCACGACCCG TCCAGGATGA AGGCCGTCAT CTACTCGAAG CACTTCTGGA GCGGCCAGGA CCGGTCGAGT TCGGCCGACA AGAGGAAGTA CGGCGACCCG GACGCTTTCC GCCCGGCCCC CGGGACCGGC CTGGTCGACA TGTCGAGGGA CAGGAACATT CCGCGCAGCC CCACCAGCCC CGGTGAGGGA TTCGTCAATT TCGACTACGG CTGGTTCGGC GCCCAGACGG AAGCGGACGC CGACAAGACC GTCTGGACCC ACGGAAATCA CTATCACGCG CCCAATGGCA GCCTTGGTGC CATGCATGTA TACGAGAGCA AGTTCCGCAA CTGGTCCGAA GGTTACTCCG ACTTCGACCG CGGAGCCTAT GTGATCACCT TCATCCCCAA GAGC (6) Preparation of library 1. Partial digestion of Streptoverticillium sp.
Sterile water was added to 60 μl of High buffer) to make a total of 594 μl and mixed.

Preheating at 37 ° C. for 5 minutes.

6 μl of BamHI (manufactured by TOYOBO) was added, mixed, and reacted at 37 ° C. for 10 minutes.

Heat treatment was performed at 65 ° C. for 15 minutes to inactivate the enzyme.

2. Cloning (EMBL3 CLO manufactured by STRATAGENE)
Use NING KIT) i) ligation ・ Precipitate 25μl of the above partially degraded DNA with ethanol,
Dissolved in 2.5 μl TE.

1.0 μl EMBL3 predigested arms (1
μg / μl), 0.5μl 10 × ligationbuffer, 0.5μl
10 mM ATP (pH 7.5), 0.5 μl T4 DNA ligase (8 units /
μl, manufactured by Boehringer Mannheim), mixed, and reacted at 4 ° C. overnight.

10 × ligation buffer 500 mM Tris-HCl, pH 7.5 70 mM MgCl ₂ 10 mM DTT ii) Packaging (GIGAPACK II GOLD PACKA manufactured by STRATAGENE)
GING EXTRACT was used) 1. An appropriate amount of the extract was transferred from a -70 ° C freezer onto dry ice, and at the same time, the sonic extract began to melt.

2. Freeze between the fingers until the furnish begins to melt /
The molten extract was warmed.

3. The above DNA solution 4 was added to the frozen / thawed extract.
μl (containing 1.6 μg) was added and placed on ice.

4. 15 μl of the sonic extract was immediately added to the frozen / thawed extract containing the DNA.

5. The mixture was stirred and mixed well without foaming.

6. Centrifuge at 4,000 g for 5 seconds and collect all contents at the bottom of the tube.

7. Incubated for 2 hours at room temperature (22 ° C.).

8. 500 μl of phage dilution buffer was added.

9. 20 μl of chloroform was added and mixed gently. 10. Centrifuged at 4,000 g for 5 seconds to sediment the debris.

11. The supernatant was collected and stored at 4 ° C.

Phage dilution buffer (per liter) 5.8 g NaCl 2.0 g MgSO ₄ 50 ml 1 M Tris-HCl, pH 7.5 5 ml 2% gelatin autoclave iii) plating 1. E. coli P2392 was inoculated into TB medium.

The properties of P2392 were hsd R514 (rk-, mk +) sup E
44, sup F58, lacY1, orΔ (lac IZY), gal K2, gal T2
2, met B1, trp R55, (P2) .The composition of TB medium is 5g
/ L NaCl, -10 g / l Bactotryptone.

2. P2392 was shake-cultured at 37 ° C. in a TB medium.

3. At an OD ₆₀₀ = 0.5, the cells were centrifuged (1,
(100 g, 15 minutes, room temperature) and suspended in 10 mM MgSO ₄ to an OD ₆₀₀ = 0.5.

4. A small amount of the supernatant of (ii) -11 was added and incubated at 37 ° C. for 15 minutes.

5. Melted in advance and warmed
top agarose (NaCl 5g / l, MgSO ₄ · H ₂ O 2g / l, Ye
ast Extract 5g / l, NZ Amine 10g / l, Agarose 0.7
%) Was mixed with 8 ml and layered on an NZY plate. (NZY
Plate: NaCl 5 g / l, MgSO ₄ .H ₂ O 2 g / l, Y
east Ext 5g / l, NZ Amine 10g / l, Agar 15g
/ L).

6. Incubated at 37 ° C. overnight.

As a result, a library consisting of 3.0 × 10 ⁴ independent clones was prepared.

(7) Screening of BTG gene Using the library obtained in (6), the BTG gene was cloned. (6) Phages in the library were plated by the method shown in (iii). After overnight culture at 37 ° C., plaques were formed and phage lifting was performed. The phage lifting was performed as follows.

1. Plates were placed at 4 ° C. for several hours.

2. A nitrocellulose filter was adhered to the plate surface (top agarose surface) (nitrocellulose filter (S & S)).

3. It was left as it was for about 2 minutes.

4. The nitrocellulose filter was peeled off and immersed in 0.5 M NaOH-1.5 M NaCl for 1 minute.

5. Filter the nitrocellulose filter with 1.5 M Na
It was immersed in Cl-1M Tris-HCl (pH 7.5) for 5 minutes.

6. Using a nitrocellulose filter with 2 × SSC
(1 × SSC = 0.15M NaCl 0.015M Na-Citrate, pH7.0)
Soaked for 0 seconds.

7. Air dried nitrocellulose on 3MM paper.

8. Insert between 3MM papers and bake at 80 ° C for 2 hours
went ing.

After lifting, pUC 19 BTG
Using a 650 bp fragment obtained by digestion with EcoRI and HindIII labeled with ³² P as a probe,
Hybridization was performed. This EcoRI, Hind
The 650 bp fragment obtained by digestion in III
It completely contains the DNA fragment amplified by R. The label of ³² P of the fragment is a multi-prime DNA.
This was performed using a label set (Amersham). The specific activity of the probe was 3 × 10 ⁸ cpm / μg-DNA. Hybridization conditions were as follows.

Prehybridization: hybridization solution (50% formamide, 1 × Denhardt ')
s (0.02% BSA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 0.1% SDS, 50 mM sodium phosphate buffer (pH
6.5), 200 μg / μl denatured salmon testis DNA) in 42 ° C.
Incubated overnight.

Hybridization: In the hybridization solution, the probe concentration was 2 ng / ml, and
Incubated overnight at 0 ° C.

Washing 1. 2 × SSC, 0.1% SDS 5 minutes × 3 times, room temperature 2.1 × SSC, 0.1% SDS 1 hour × 2 times, 68 ° C. After washing, air-dry the nitrocellulose filter, and autoradiogram Was taken.

By the above series of operations, about 40,000 plaques were initially screened (first screening). As a result, 16 clones showing a strong signal were obtained. Further screening was performed on these 16 clones to obtain these clones as single plaques. As a result, six single-plaque clones showing a strong signal were obtained.

(8) Structural Analysis of Clone DNA In order to examine the DNA structure of the obtained six clones, DNA was prepared from the six clones. For DNA preparation, refer to “Molecular, Cloning, a laboratory manual 2n
d Edition ”.

From the DNA thus obtained, restriction enzymes BamHI, SphI, NcoI, BglII, KpnI (all of which are TOYOB
O) was used to generate a restriction map. As a result, these clones contain almost identical DNA fragments,
The restriction map was determined as shown in FIG.

Next, the DNA was cleaved with each restriction enzyme, electrophoresed, the DNA was fixed on a nitrocellulose filter, and Southern hybridization was carried out using the probe previously used for screening the BTG gene. Hybridization conditions were the same as those used in the BTG gene screening.

As a result, it was found that the NcoI 3.6 kbp fragment on the map hybridized to the probe and gave a strong signal. Therefore, at least BTG
Part of the gene was thought to be in the NcoI fragment.

(9) Subcloning of NcoI 3.6 kbp Fragment Next, the NcoI 3.6 kbp fragment was subcloned into a plasmid. DNA of the obtained phage clone
Was digested with NcoI, and a 3.6 kbp DNA fragment was recovered using low melting point agarose. This NcoI fragment was transferred to plasmid pTV.
118N (Takara Shuzo) was ligated with DNA cut with NcoI, and Escherichia coli DH5α was transformed with this DNA.
From the obtained transformant, a plasmid pTV118 NcoI in which the NcoI 3.6 kbp fragment was subcloned into pTV118N was obtained.

(10) DNA sequence of BTG gene DNA sequence was performed using the obtained pTV118 NcoI as a template. As the method, a method similar to the method described in the section (4) was used. However, here, the reaction temperature was set to 48 ° C., and depending on the degree of sequence compression, SSB (Single Stranded DNA Binding Prot.
ein; TOYOBO).

The base sequence determined is shown in Table 6 below (SEQ ID NO: 3).

[0109]

[Table 15] Table of _{6 TGCGGCGACG CGTAGGCAAT GGGGGTTCAT CGCGACGTGC TTCCGCACGG CCGCGTTCAA 1} CGATGTTCCA CGACAAAGGA GTTGCAGGTT TCCATGCGCT ATACGCCGGA GGCTCTCGTC TTCGCCACTA TGAGTGCGGT TTATGCACCG CCGGATTCAT GCCGTCGGCC GGCGAGGCCG CCGCCGACAA TGGCGCGGGG GAAGAGACGA AGTCCTACGC CGAAACCTAC CGCCTCACGG CGGATGACGT CGCGACATCA ACGCGCTCAA CGAAGCGCTC CGGCCGCTTC GAGCGCCGGC CCGTCGTTCC GGGCCCCCGA CTCCGACGAC AGGGTCACCC CTCCCGCCGA GCCGCTCGAC AGGATGCCCG ACCCGTACCG TCCCTCGTAC GGCAGGGCCG AGACGGTCGT CAACAACTAC ATACGCAAGT GGCAGCAGGT CTACAGCCAC CGCGACGGCA GGAAGCAGCA GATGACCGAG GAGCAACGGG AGTGGCTGTC CTACGGCTGC GTCGGTGTCA CCTGGGTCAA TTCGGGTCAG TACCCCACGA ACAGACTGGC CTTCGCGTCC TTCGACGAGG ACAGGTTCAA GAACGAGCTG AAGAACGGCA GGCCCCGGTC CGGCGAGACG CGGGCGGAGT TCGAGGGCCG CGTCGCGAAG GAGAGCTTTG ATGAAGAGAA GGGGTTCCAG CGGGCGCGTG AGGTGGCGTC CGTGATGAAC AGGGCCCTGG AGAACGCCCA CGACGAGAGC GCTTACCTCG ACAACCTCAA GAAGGAACTG GCGAACGGCA ACGACGCCCT GCGCAACGAG GACGCCCGTT CCCCGTTCTA CTCGGCGCTG CGGAACACGC CGTCCTTTAA GGAGCGGAAC GGAGGCAATC ACG ACCCGTC CAGGATGAAG GCCGTCATCT ACTCGAAGCA CTTCTGGAGC GGCCAGGACC GGTCGAGTTC GGCCGACAAG AGGAAGTACG GCGACCCGGA CGCTTTCCGC CCGGCCCCCG GGACCGGCCT GGTCGACATG TCGAGGGACA GGAACATTCC GCGCAGCCCC ACCAGCCCCG GTGAGGGATT CGTCAATTTC GACTACGGCT GGTTCGGCGC CCAGACGGAA GCGGACGCCG ACAAGACCGT CTGGACCCAC GGAAATCACT ATCACGCGCC CAATGGCAGC CTTGGTGCCA TGCATGTATA CGAGAGCAAG TTCCGCAACT GGTCCGAAGG TTACTCCGAC TTCGACCGCG GAGCCTATGT GATCACCTTC 1218 ATCCCCAAGA GCTGGAACAC CGCCCCCGAC AAGGTAAAGC AGGGCTGGCC GTGATGTGAG BTG than ^CG nucleotide sequence The start codon of the gene is ATG at position 1.
It was estimated. The reason is that (a) a stop codon is present at 81b upstream of position 1 and the open reading frame of the BTG gene is thought to start from downstream of this position; (b) a typical 13 b upstream of ATG at position 1 Na SD
The sequence (5'-AAAGGAG-3 ') is present; (c) the region about 20 amino acids from the methionine of the ATG at position 1 is a relatively hydrophobic portion, having a signal sequence-like sequence: This is from three points.

A stop codon is present at position 1219 at the 3 'end, and the C terminal of BTG is considered to be proline at position 1216. BTG determined by amino acid sequence in open reading frame determined from nucleotide sequence
Of the N-terminal amino acid is aspartic acid at position 226. Therefore, it was found that the BTG gene open reading frame obtained here had a structure obtained by adding 75 amino acids to the structure obtained from the amino acid sequence.

Example 2 Chemical Synthesis of BTG Gene Design and Synthesis of BTG Gene As described above, the DNA of the BTG gene was designed based on the entire amino acid sequence of the BTG thus clarified. At that time, the codon usage of E. coli and yeast (Ikemura, T. and Ozeki, H.,
Cold Spring Harbor Symp.Quant.Biol., 47 , 1087 (19
In consideration of 83)), a recognition site of a restriction enzyme used for assembling and linking the fragments was arranged on the DNA chains at both ends of each of the fragments (1) to (5).

[0112]

[Table 16] Next, the designed gene DNA was chemically synthesized by a phosphoramidite method using a DNA synthesizer 380A manufactured by Applied Biosystems.

Judging from the reliability and operability of current DNA synthesizers and DNA purification methods, the number of DNA strands per
It was about 40 bases. First, the BTG is 331 amino acids,
That is, a gene of 993 bases is required at least. Further, since it is necessary to incorporate the DNA into a plasmid as a double strand, it is necessary to synthesize twice as much DNA. Actually, since a stop codon was introduced and a restriction enzyme recognition site for ligation of each fragment was also required, a total of 54 DNA strands were synthesized on 27 front and back sides. In addition, since it is necessary to confirm the nucleotide sequence at the time of incorporation into the plasmid, it is easier to integrate the plasmid into lengths (about 200 bp) into which the nucleotide sequence can be reliably determined.

Therefore, instead of assembling the entire gene at once, the first fragment was assembled into five blocks of about 200 bp each, and the base sequence was confirmed there before the whole was constructed.

Construction of BTG Gene (Synthetic Type) First, each fragment (about 200 bp) divided into 5 blocks
Was constructed. Since there is no phosphate at the 5 'end of the oligonucleotide obtained by chemical synthesis,
Phosphate was attached to the 5 'end using polynucleotide kinase. At that time, phosphorylation of the DNA strands located at both 5 'ends of each fragment was performed after ligation.

Each oligonucleotide 100 pmole (in water) 7.5 μl 10x buffer * 1 μl 10 mM ATP 1 μl polynucleotide kinase (Takara Shuzo) 0.5 μl (5 units) 10 μl * 10x buffer 650 mM Tris-HCl (pH 7.6) 100 mM MgCl ₂ 150 mM dithio Threitol 10 mM spermidine The above composition was placed in an Eppendorf tube, reacted at 37 ° C. for 1 hour, and then boiled for 3 minutes to inactivate the enzyme.
Next, 5 μl of complementary pairs are mixed (total 10 μl).
1) After boiling in a hot water bath at 90 ° C. for 3 minutes, annealing was performed by naturally lowering the temperature of the hot water to 37 ° C. After that, two pairs of DNAs (20 μl) were ligated next to each other using ligase.

DNA 2 pairs 20 μl 150 mM dithiothreitol (DTT) 2 μl 10 mM ATP 2 μl ligase (Takara Shuzo) 0.5 μl (150 units) 24.5 μl The above composition was placed in an Eppendorf tube, and allowed to react at 16 ° C. for 30 minutes. Furthermore, in order to connect the blocks next to each other and connect the blocks, 22 μl of each of the above-mentioned reaction solutions was added to 3 portions.
This mixture was mixed (total 66 μl), and ligase 0.5 μl was added. After reaction at 16 ° C. for 30 minutes, 10% polyacrylamide gel electrophoresis was performed, and a DNA fragment of about 200 bp was recovered from the gel.

The thus obtained DNA fragments (1) to
(5) has an EcoRI recognition site at the 5 'end and Hind at the 3' end.
III There is a recognition site. Next, after phosphorylation of both 5 'ends of each fragment, an appropriate amount was
Plasmid pUC18 digested with HindIII (Yanisch-Pe
rron, C. et al., Gene, 33 , 103 (1985)), and a ligation reaction was performed at 16 ° C. for 1 hour using a ligation kit (Takara Shuzo).

Next, each mixture was used to transform E. coli strain JM109 (re
cA1, Δlacpro, endA1, gryA96, thi-1, hsdR17, supE4
^{4, relA1, λ -, (} F'traD36, proAB, lacI q Z ΔM15))
Was introduced.

The E. coli JM109 strain is a pUC plasmid DNA.
When transforming M13 or transducing M13 phage vector DNA, it is easy to select recombinants by using β-galactosidase activity recovery by lacZα peptide generated from vector DNA and lacZΔM15 encoded by JM109F ′. Strain. That is, JM109 containing plasmid pUC18 in a medium containing IPTG (isopropyl-β-D-thiogalactopyranoside) and X-Gal (5-bromo-4-chloro-3-indole-β-D-galactoside).
The strain appears as a blue colony showing β-galactosidase activity, while the JM109 strain carrying the recombinant plasmid into which the foreign DNA fragment has been inserted does not recover the β-galactosidase activity and appears as a colorless colony.

From some colorless colonies, plasmids were prepared and their DNA sequenced (Sa
nger, F. et al., J. Mol. Biol., 143 , 161 (1980)), and clones having the correct nucleotide sequence as designed for each inserted fragment were selected.

Further, each fragment was ligated using each restriction enzyme recognition site.

First, the connection of fragments (2) and (3)
Using the ScaI recognition site present in the ampicillin resistance gene of C18, fragments (2) and (3) were replaced with ScaI and XbaI
, And using a ScaI.XbaI fragment containing the BTG gene. In the same manner, fragments (4) and (5) were ligated by digesting fragments (4) and (5) with ScaI and NcoI, followed by BT
This was performed using a ScaI / NcoI fragment containing the G gene. Further, the ligation of fragments (2) (3) (4) (5) was similarly performed by cutting the fragments (2) (3) and (4) (5) with ScaI and BglII, • Performed using the Bgl II fragment.

Finally, EcoRI / HpaI of fragment (1) was used.
HpaI / Hind of aggregate of fragment and fragment (2) (3) (4) (5)
The high expression secretion vector pIN-III-ompA2 for Escherichia coli (Ghrayeb,
J. et al., EMBO J., 3 , 2437 (1984)), and a ligation reaction was carried out at 16 ° C. for 30 minutes using a ligation kit.
Next, this mixture was used for the E. coli strain JA221 (hsdM ⁺ , trpE5, l
euB6, lacY, recA / F ' , lacI q, lac +, pro +) (Nakamur
a, K. et al., J. Mol. Appl. Genet., 1 , 289 (1982)). Prepare several plasmids from the resulting colonies,
It was confirmed that the BTG gene (about 1 kb) was correctly inserted. The BTG expression and secretion vector thus obtained was
Called MPA-BTG. Table 2 shows the nucleotide sequence of the inserted chemically synthesized BTG gene.

The plasmid pIN- used in this example was used.
III -OmpA2 promoter of the outer membrane lipoprotein of E. coli (lpp ^P), and the promoter of the lactose operon,
The operator (lac ^PO ) contains the signal peptide of the outer membrane protein OmpA of Escherichia coli, and the expression of the gene integrated downstream thereof is induced by the addition of IPTG, and the gene product is accumulated in the periplasm. Gene products have been reported to accumulate in the periplasm in several cases, including subtilisin (Ikemura, H. et al.).
J. Biol. Chem., 262 , 7859 (1987), Hsiung, HM
Bio / Technology, 4 , 991 (1986), etc.). Purification of BTG protein is carried out as usual by extracting periplasmic proteins from the periplasm by the osmotic shock method (Koshland, D. et al., Cell, 20 , 749 (1980)), followed by ammonium sulfate precipitation and various ion exchange chromatography. , Gel filtration, HPLC and the like.

The plasmid used for the expression of the synthesized BTG gene is not limited to pIN-III-ompA, and other expression plasmids can be used. Those skilled in the art can easily select these depending on the expression system.

Example 3 Large Intestine of BTG Gene (Synthetic Type)
Plasmid pOMPA-BTG incorporating a synthetic BTG gene expressed in fungi
E. coli JA221 strain (FERM P-11745) carrying E. coli is incubated at 37 ° C. for 2 hours in an M9 casamino acid medium containing 50 μg / ml of ampicillin.
After shaking culture for a period of time, the culture temperature was lowered to 23 ° C, IPTG was added to 1 mM, and the mixture was further shaken at 23 ° C for 4 hours. Next, 50 ml of the culture was collected by centrifugation, and the cells were suspended in 2.5 ml of 20% sucrose and 10 mM Tris-HCl (pH 7.5).
After adding 0.125 ml of 0.5 M EDTA (pH 8.0), the mixture was left on ice for 30 minutes. After centrifugation at 12000 rpm for 10 minutes to separate the supernatant and the cells, the cell fraction was suspended in 3 ml of distilled water and left on ice for 30 minutes. After centrifugation at 12000 rpm for 10 minutes, the supernatant was separated from the cells. Next, these supernatants were combined and ultracentrifuged at 38,000 rpm for 60 minutes, and the supernatant was used as a periplasm fraction.

Further, the bacterial cell fraction was suspended in 3 ml of distilled water and subjected to ultrasonic crushing (200 W, 5 minutes) to obtain a cytoplasmic fraction.

Measurement of BTG activity of each fraction The BTG activity of each prepared fraction (culture supernatant, periplasm, cytoplasm) was measured by the hydroxamic acid method.

[0130] The measurement method is basically, J.Biol.Chem. 241
The method of Folk and Cole described in 5518 (1966) (Colorime
tric hydroxamate Procefure). That is, a reaction is carried out in the presence or absence of Ca ²⁺ using benzyloxycarbonyl-L-glutamylglycine (CBZ-gln-gly) and hydroxylamine as substrates,
In this method, the produced hydroxamic acid is formed as an iron complex in the presence of trichloroacetic acid, the absorption at 525 nm is measured, and the amount of hydroxamic acid is determined from a calibration curve to calculate the activity.

<Activity measurement method> Reagent A 0.2 M Tris-HCl buffer (pH 6.0) 0.1 M hydroxylamine 0.05 M Calcium chloride 0.01 M Reduced glutathione 0.03 M CBZ-gln-gly (manufactured by Kokusan Chemical) Reagent B 3N hydrochloric acid 1 12% vol - were mixed with 0.4ml reagent a 0.4ml of trichloroacetic acid 1 volume _{5% FeCl 3 · 6H 2 O} ( dissolved in 0.1 N-HCl) 1 volume sample (enzyme solution)
After reaction at 37 ° C. for 10 minutes, 0.4 ml of reagent B was added to terminate the reaction and form an iron complex, and then the absorbance at 525 nm was measured.

As a control, the absorbance of an enzyme solution which had been reacted in advance using an enzyme solution which had been heat-inactivated in advance was measured to determine the difference in absorbance from the enzyme solution, and L-glutamic acid γ-monosaccharide was separately used instead of the enzyme solution. A calibration curve was prepared using hydroxamic acid, the amount of generated hydroxamic acid was determined from the absorbance, and the enzyme activity for producing 1 micromol of hydroxamic acid per minute was defined as 1 unit.

The measurement results are shown below.

[0134]

[Table 17] When induced by IPTG to any fraction, BT is slightly weak
G activity could be detected.

Further, in order to confirm that a BTG gene product was produced, Western blotting with an anti-BTG antibody prepared in rabbits using purified BTG was performed.
This was performed using Vectastain ABC kit from Vector.

That is, 0.5 μl of the protein from each fraction was subjected to SDS-polyacrylamide gel electrophoresis, and the gel after the electrophoresis was transferred to a membrane (Immobilon; Millipore) to bind the protein serving as an antigen to the membrane. Thereafter, a rabbit anti-BTG antibody (antibody titer 64
Western blotting) was performed, and the gene product was confirmed at the protein level.

As a result, in the following lanes 1, 3, 5 and 7, colored bands appeared at the same positions, and the protein showing a reaction with the anti-BTG antibody was the same as that of purified BTG in any of the IPTG-added fractions. It was detected at the position of the molecular weight.

1: Purified BTG (control) 2: Culture supernatant (no IPTG added) 3: Same as above (IPTG 1 mM added) 4: Periplasm (No IPTG added) 5: Same as above (IPTG 1 mM added) 6: Cytoplasm (No IPTG added) Example 7: Expression of BTG gene (natural type) in actinomycetes (1) Acquisition of plasmid vector (pIJ702) Streptomyces lividans 3131 containing pIJ702 (ATCC 3528)
7) was cultured under the following medium conditions at 30 ° C. for 2 days.

[YEM medium + 0.5% glycine + 50 μg / ml thiostrepton] 0.3% yeast extract 0.5% peptone 0.3% malt extract 0.1% magnesium chloride 1.0% glucose 0% saccharose 0.5% glycine 0.1% 50 mg / ml thiostrepton solution (Sigma: dimethyl sulfoxide solution) / l (pH 7.0) 200 ml of culture medium cultured under the above conditions was centrifuged (1
2,000 g, 4 ° C, 10 minutes), and the resulting precipitate (cells)
Was washed with 50 mM Tris-HCl (pH 8.0) -5 mM EDTA-50 mM NaCl. After washing, the cells obtained by centrifugation (1,300 g, room temperature, 5 minutes) were subjected to 50 mM Tris-HCl (pH 8.0) -10 mM EDTA-25.
% Sucrose (hereinafter referred to as “TE-Sucrose”) in 10 ml. Next contains 30mg / ml lysozyme (Sigma)
2 ml of TE-Sucrose and 4 ml of 0.25M EDTA were added, and this was added to 3 ml.
Incubated at 7 ° C. for 30 minutes. After the incubation, 2 ml of 20% SDS was added, 5 ml of 5M NaCl was further added, and the mixture was gently stirred, followed by incubation at 0 ° C. overnight. Next, 30% polyethylene glycol 6000 was added to the supernatant obtained by centrifugation (100,000 g, 4 ° C., 40 minutes) to a final concentration of 10%, and the mixture was incubated at 0 ° C. for 4.5 hours. Thereafter, the mixture was centrifuged (900 g, 4 ° C., 5 minutes), and the precipitate was washed with 10 mM Tris-HCl (pH 8.0) -1 mM EDTA-50 mM.
Dissolved in NaCl. And 16.8 g of cesium chloride and 10
Ethidium bromide to 10 mM Tris-HCl to a concentration of mg / ml
(pH 8.0) -1 mM EDTA (hereinafter referred to as "TE") dissolved in 1.2 ml, and the residue was removed by centrifugation (1,300 g, room temperature, 15 minutes), followed by centrifugation (230,000 g). , 20 ° C, 12 hours). After centrifugation, a plasmid DNA layer was obtained under ultraviolet irradiation. Then T
Extraction with butanol saturated with E was performed to remove ethidium bromide. This extraction was repeated three times. The obtained product was dialyzed overnight at 4 ° C. with TE. Thereafter, extraction was performed once with TE-saturated phenol and twice with chloroform / isoamyl alcohol. Next, 1/10
A volume of 3M sodium acetate (pH 5.2) solution and 2 volumes of ethanol were added, and the mixture was allowed to stand at -80 ° C for 30 minutes. Thereafter, the precipitate was recovered by centrifugation (12,000 g, 4 ° C., 15 minutes), and the precipitate was washed with 70% ethanol and dried. This was dissolved in 200 μl of TE. The amount of DNA finally obtained was about 10 μg.

(2) Obtaining a Host Streptomyces lividans 3131 containing pIJ702 was cultured in a YEME medium at 30 ° C. for 7 days.

Next, the culture solution was added to YEME medium at 10 ⁵ to 1
Diluted ^0-9 fold, Y of each dilution by 100μl
The seeds were spread on five EME agar media (plate agar media obtained by adding 1.5% agar to YEME media). Then, the cells were cultured at 30 ° C. for one week. After the culture, RepliPlate ^TM Colony Transfer P
ad (Takara Shuzo), and replicated on a YME agar medium containing 200 μg / ml thiostrepton.
C. for 1 week. One thiostrepton-sensitive strain thus isolated was named Streptomyces lividans 3131-TS and used as a host.

(3) Preparation of Streptomyces lividans 3131-TS Protoplasts Streptomyces lividans 3131-TS obtained in (2) was converted to Y
The cells were cultured in EME medium + 0.5% glycine at 30 ° C. for 2 days. Centrifuge 200 ml of culture solution (1,300 g, room temperature, 10
The resulting cells were suspended in 72 ml of 0.35M saccharose. Next, this suspension was centrifuged (1,300 g, room temperature, 10 minutes), and the cells were resuspended in 60 ml of a P buffer (described below) containing 1 mg / ml lysozyme (Sigma). , 2.5 hours. After the incubation, this suspension was filtered with absorbent cotton to remove the residue.
Next, the obtained filtrate was centrifuged (1,300 g, room temperature, 10
Min) and washed with 25 ml of P buffer. After repeating this washing twice, the precipitate was suspended in 1 ml of P buffer to obtain a protoplast suspension.

[P buffer] TES [N-Tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid] 5.73 g Saccharose 103 g Magnesium chloride 2.03 g Potassium sulfate 0.5 g Calcium chloride 3.68 g Trace element solution 2 ml / l (PH 7.4) A 1% monopotassium phosphate solution was separately prepared, and 1 ml per 100 ml P buffer was added immediately before use.

[Trace element solution] Zinc chloride 40 mg Ferric chloride 200 mg Cupric chloride 10 mg Manganese chloride 10 mg Sodium tetraborate 10 mg Ammonium molybdate 10 mg / l (4) Expression type BTG gene fragment in actinomycetes (Mp-BTG)
Construction of spm) The BTG gene was expected to form a prepro-form as a result of DNA sequencing. Therefore, Streptoverticillium sp.
We thought that the expression in ptomyces lividans 3131-TS would facilitate the processing of the precursor to the mature form more smoothly than in the case of other microorganisms. Therefore, an expression type BTG gene was constructed as follows.

I) Obtaining a fragment containing a BTG gene signal, a pro region and a structural gene (hereinafter referred to as “BTG spm”) using a 3.6 kbp BTG gene NcoI fragment as a template by PCR. Fragments ").

Primer # 3 and Primer # 4 used for PCR
Is shown below.

[0147]

[Table 18]

The detailed conditions of the PCR method are described in Example 1.
Is the same as

Ii) Obtaining a Mel (Melanin Synthetic Gene) Promoter Region Fragment The mel gene was selected as the BTG gene expression promoter region. Therefore, a fragment of the promoter region of the mel gene (hereinafter referred to as “Mp fragment”) was obtained by PCR using pIJ702 as a template.

Primer # 5 and Primer # 6 used for PCR
Is shown below.

[0151]

[Table 19]

The detailed conditions of the PCR method are described in Example 1.
Is the same as

Iii) Construction of BTG gene fragment (Mp-BTG spm) The Mp fragment amplified by PCR and pUC19 were individually
After cutting with RI and SacI (TOYOBO), the target size was recovered using low melting point agarose. Next, these were ligated, and E. coli DH5α was transformed with X-gal and IPTG.
It was transformed in the presence. Then, the Mp fragment obtained from the ampicillin-resistant white colony was pUC19 EcoRI-SacI.
The plasmid integrated into the site was designated as pUC19-Mp and used for the following experiments.

Next, BTG spm similarly amplified by PCR
The fragment and pUC19-Mp were treated with BamHI and SacI (TOYOBO), respectively, and subcloned. BT obtained in this way
The plasmid in which the G spm fragment was incorporated into the BamHI-SacI site of pUC19-Mp was used as pUC19-Mp-BTG spm in the following experiments.

The obtained plasmid pUC19-Mp-BTG spm is Ps
The mixture was treated with tI and BglII (TOYOBO) to obtain a 1.4 kbp Mp-BTG spm fragment.

For ligation, a ligation kit (Takara Shuzo) was used.

(5) Streptomyces livid of BTG gene
Introduction to ans 3131-TS 1.4kbp Mp-BTG spm fragment obtained in the previous section and 5.1kbp pIJ70
2 The Pst-BglII fragment was ligated. For the ligation, the following reaction solution was prepared and incubated at 4 ° C. overnight.

8.5 kl of 1.4 kbp Mp-BTG spm fragment (about 500 ng) 5.1 kbp pIJ702 PstI-BglII fragment 8.5 μl (about 500 ng) 5 units / μl T4 DNA ligase (Boehringer) 1.0 μl 10x ligation buffer (Boehringer) 2.0 μl After incubation, Streptomyces lividan
s 3131-TS was transformed. Transformation was performed as follows.

1. Prepare the following reaction mixture, total volume 140μ
l.

DNA solution 20 μl Streptomyces lividans 3131-TS protoplast 100 μl 0.35 M saccharose 20 μl 2. 1.5 ml of P buffer containing 20% polyethylene glycol 1000 was added and gently mixed by pipetting.

[0161] 3. It was left at room temperature for 2 minutes.

4. The pellet was collected by centrifugation (1,700 g, room temperature, 10 minutes).

5. The resulting pellet was washed with P buffer. This operation was repeated twice.

6. After resuspending the pellet in 1 ml of P buffer, 200 μl was applied to R-2 medium.

[R-2 Medium] R-2 / A and R-2 / B shown below were separately prepared, and R-2 / A-2
/ A, a mixture of R-2 / B, was further mixed with 1% KH ₂ PO ₄ in a ratio of final volume 200ml per 1 ml.

R-2 / A Potassium sulfate 0.5 g Magnesium chloride 20.2 g Calcium chloride 5.9 g Glucose 20.0 g Proline 6.0 g Casamino acid 0.2 g Trace element solution 4 ml Agar 44.0 g / l R- 2 / B TES 11.5 g Yeast extract 10.0 g Saccharose 203 g / l (pH 7.4) 7. Incubated at 30 ° C. for 18 hours.

8. 1 ml of P buffer containing 200 μg / ml thiostrepton and 400 μg / ml tyrosine was added to cover the entire surface of the agar.

9. Further, after incubation at 30 ° C. for 7 days, thiostrepton-resistant white colonies were obtained.
Those retaining pIJ702 into which no foreign DNA fragment has been inserted synthesize melanin by the mel gene and appear as black colonies. Plasmids were prepared from several white colonies thus obtained, and it was confirmed whether the desired BTG gene was inserted.

The obtained BTG expression secretion vector was
It was named pIJ702-BTG.

(6) Expression of BTG gene An actinomycete transformed with pIJ702-BTG incorporating the BTG gene was cultured as Streptomyceslividans AKW-1 at 30 ° C. for 5 days under the following medium conditions.

Polypeptone 2% Soluble starch 2% Yeast extract 0.2% Dipotassium phosphate 0.2% Magnesium sulfate 0.1% Adecanol LG 126 0.05% 50 mg / ml Thiostrepton solution 0.1% Above 100 ml of the culture medium cultured under the conditions was centrifuged (1.
(2,000 g, 4 ° C., 10 minutes), and the obtained supernatant was concentrated approximately 17-fold using an ultrafiltration membrane (Amicon) having a molecular weight cut off of 1,000.

Next, the prepared sample was purified BT
EIA with anti-BTG antibody prepared in rabbits using G
Quantified by the method. As a result, about 0.1 mg / l of BTG could be detected.

The EIA was performed as follows.

1. 500 μl of buffer A in 50 μl of sample
Was added and mixed, and one antibody-bound bead (beads used was Sekisui polystyrene # 80) was added thereto.
Incubated at 37 ° C for 30 minutes.

[0175] 2. The reaction solution was removed, and the beads were washed twice with 10 mM phosphate buffer (pH 7.0).

[0176] 3. 500 μl of the β-galactosidase-labeled antibody (50 mu / 500 μl buffer A) was added to the washed beads.
Was added and incubated at 37 ° C. for 30 minutes.

[0177] 4. The reaction solution was removed, and the beads were washed twice with 10 mM phosphate buffer (pH 7.0).

[0178] 5. Washed beads with CPRG solution 500
μl and incubated at 37 ° C. for 30 minutes.

6. After adding 2 ml of the reaction stop solution, 575 nm
Was measured for absorbance.

[Buffer A] 0.1 M sodium chloride 0.1% BSA 1 mM magnesium chloride 0.1% sodium azide 10 mM phosphate buffer (pH 7.0) [CPRG solution] CPRG (chlorophenol red β-D- (Galactopyranoside, Boehringer) 1 g BSA 333.2 mg monopotassium phosphate 833.2 mg sodium sulfite 166.8 mg GH 333.2 ml / l (pH 5.0) [GH] 5% hydrolyzed gelatin 0.3 M sodium chloride 1 mM magnesium chloride 0.1 % Sodium azide 0.1% BSA 50 mM phosphate buffer (pH 7.0) [Reaction stop solution] 10 mM disodium ethylenediaminetetraacetate 1% galactose 0.1% sodium azide 50 mM phosphate buffer (pH 7.0) Recombinant BTG was analyzed by Western blot. First, the sample is processed by the existing method,
SDS-PAGE electrophoresis was performed. SDS-PAG
After electrophoresis, the proteins on the gel are separated by Electrophoretic
Transfer membrane (Immobilon) using Transfer Kit (LKB)
^TM (Millipore)). Transfer conditions were in accordance with the instructions attached to the Electrophoretic Transfer Kit. After the transfer, the membrane was incubated for 1 hour in 10 ml of Blocking Buffer (20 mM Tris-HCl (pH 7.9), 5% skim milk). Thereafter, the membrane was rinsed with BTG antiserum 200 times
Buffer (10 mM Tris-HCl (pH 7.9), 0.15 M NaCl, 0.1 mM ED
Incubated overnight at 4 ° C. in 10 ml of a solution diluted with TA (3Na), 0.25% skim milk, 0.01% NaN ₃ ).
After incubation, the solution is discarded and the membrane is reconstituted in 20 ml.
After washing twice with Rinse Buffer, the membrane is
i rabbit ¹²⁵ I label whole antibody (Amersham) 1
The cells were transferred to 10 ml of RinseBuffer containing μCi and incubated at 4 ° C. for 4 hours. After incubation, the membrane was washed twice with 20 ml of Rinse Buffer, after which the membrane was removed and air-dried. After air drying, the membrane was subjected to autoradiography to detect a signal.

As a result, BTG as a mature protein was observed at a position of about 37 KDa having the same molecular weight as purified BTG (see FIG. 2). This is the Streptomyces liv
In BTG gene expression by idans AKW-1, this indicates that the mature protein was secreted despite the introduction of the precursor gene, suggesting that correct processing was performed.

Example 5: BTG gene (synthetic type) yeast
The expression of BTG in yeast was carried out in two ways, using a mature BTG gene or an expression vector having a BTG prosequence gene and a mature BTG gene incorporated downstream of the signal sequence gene.

As an expression vector, an Escherichia coli / yeast shuttle vector pNJ 1053 containing a promoter sequence of a glycolytic enolase gene ( ENO1 ) known to have strong promoter activity in yeast was used. E
Expression in yeast using the promoter of NO1 was performed using human lysozyme (Ichikawa, K. et al . , Agric. Biol. Chem. , 53 ,
2687 (1989)), but this vector is pBR32.
Replication origin at 2 from E. coli, the ampicillin resistance gene and yeast 2μm origin of replication, the high copy type having both a LEU2 gene as a selection marker (YE
p) Vector.

Here, as the signal sequence, a human gastrin signal sequence (Kato, K. et al., Gene , 26 , 53-57 (1)
983)). This signal sequence consisting of 21 amino acid residues is one in which the carboxyl terminal side of alanine at position 21 is cleaved by signal peptidase, and is used for secretion of heterologous proteins in yeast including human-α-amylase (Sato, T. et al., Gene , 83, 355-365
(1989)).

It is also believed that the prosequence intervening between the signal sequence and the mature gene sequence plays an important role in expressing the activity of the translated protein as is known in Bacillus subtilis (Ikemura). , H. et al . , J. Biol. C
hem. , 262 , 7859-7864 (1987)). The BTG pro sequence used in this example refers to 39 amino acid residues from the lysine at position −39 to the proline at position −1 which exist downstream of the BTG signal-like sequence identified in Example 1.

(1) BTG matured body expression secretion vector pN
Construction of J1053-BTG First, a vector having a gastrin signal sequence (Sat
o, T., et al., Gene , 83 , 355-365 (1989)). An XhoI / HindIII fragment of about 90 bp containing a gene encoding a signal sequence was cut out, and a vector pHSG396 (Takara Shuzo, Takeshita) previously digested with XhoI and HindIII was used. , S. et al., Gen
e , 61 , 63-74 (1987)) to obtain pHSG396-GS.
The HindIII digest was ligated to a synthetic oligonucleotide of about 30 bp shown below and a PvuII-HindIII fragment (about 1 kb) of the BTG gene chemically synthesized in Example 2, and BT
Those in which the G mature gene was correctly inserted downstream of the signal sequence gene were selected.

[0187]

5′- AGCTT GGGAT TCTGATGACA GAGTCACTCC AC CAG -3 ′ 3′- A CCCTA AGACTACTGT CTCAGTGAGG TG GTC -5 ′ HindIII PvuII Further, SalI and Hind present upstream of the XhoI recognition site
Cleavage with III yields a fragment of approximately 1.1 kbp, yeast expression vector
Sal present downstream of the ENO1 promoter of pNJ1053
Inserted between I and HindIII. This was introduced into E. coli JM109 strain to obtain an expression secretion vector pNJ1053-BTG.

(2) BTG prosequence-mature expression
Construction of the vector pNJ1053-proBTG First, a gene encoding the BTG pro sequence was synthesized. At that time, considering the codon usage of E. coli and yeast,
Restriction enzyme recognition sites for ligation with signal sequence genes and mature genes were also arranged. That is, Hi at the 5 'end of the prosequence gene for ligation to the signal sequence gene.
At the 3 'end, a sequence for linking to the PvuII recognition site of the BTG gene chemically synthesized in Example 2 was arranged at the 3' end.

[0189]

[Table 21]

For the synthesis of this sequence, three DNA strands of about 50 bases per one were chemically synthesized in total on three sides. (DN
For A synthesis, a DNA synthesizer 380A manufactured by Applied Biosystems was used. ) The chemically synthesized oligonucleotide was subjected to a reaction such as phosphorylation, annealing and ligation in the same procedure as in the synthesis of the BTG gene chemically synthesized in Example 2. After the reaction, 10% polyacrylamide gel electrophoresis was performed, and the D
The NA fragment was recovered from the gel. Next, as in (1), p
The HindIII digest of HSG396-GS was ligated to the recovered synthetic oligonucleotide of about 150 bp and the PvuII / HindIII fragment (about 1 kb) of the BTG gene chemically synthesized in Example 2, and the BTG pro sequence gene was linked to the signal sequence. Those that were correctly inserted between the BTG mature genes were selected. Furthermore, SalI and HindIII existing upstream of the XhoI recognition site
To obtain a fragment of about 1.2 kbp.
SalI located downstream of the ENO1 promoter of NJ1053 ,
Inserted between HindIII. This was introduced into E. coli JM109 strain to obtain an expression secretion vector pNJ1053-proBTG.

(3) Expression of BTG gene in yeast Expression secretion vector pNJ105 obtained in each of (1) and (2)
3-BTG and pNJ1053-proBTG host yeast Saccharomyces
cerevisiae KSC22-1C (MATa, ssl1, leu2 , his -, ur
a3 ) is introduced by the alkali metal treatment method (Ito, H. et al., J.
Bacteriol , 153 , 163-168 (1983)). At this time, a minimal medium is required for selection and culture of clones transformed with a plasmid incorporating a gene complementing the auxotrophic mutation, but the minimum medium used for selection of transformants was obtained from Difco. Commercially available bacto yeast nitrog
enbase (YNB) with 2% glucose and 20 mg / l L-histidine hydrochloride, 20 mg / l uracil depending on the nutritional requirements of the host yeast (additionally 2 mg / l uracil for plates). % Agar). Leu
^The transformant which became ⁺ formed a colony in 2 to 4 days when cultured at 30 ° C.

Each of the transformants carrying these secretion expression vectors pNJ1053-BTG or pNJ1053-proBTG was pre-incubated overnight at 30 ° C. in a minimal medium to prevent the plasmid from dropping out, and then 10 ml of a synthetic medium having the composition shown below. (5%) and cultured with shaking at 30 ° C. for 2 days.

Synthetic medium 0.67% YNB 8% Glucose 20mg / l L-Histidine hydrochloride 20mg / l Uracil 0.5% Casamino acid (Difco) followed by the glass bead method (Hitzeman, RA and other Science ,
219 , 620-625 (1983)). That is, cells are collected from 10 ml of the culture solution by centrifugation,
After washing once with sterile water, the cells are suspended in 1 ml of Tris-HCl (pH 6.0). Add 1 ml glass beads (B. Brown, diameter
0.45-0.5 mm) and vigorously stirred three times for 1 minute at 0-4 ° C. using a Vortex mixer. After separating the glass beads by low-speed centrifugation, the supernatant was transferred to an Eppendorf tube, and further centrifuged at 12,000 rpm for 5 minutes, and the supernatant was taken as an extract.

Therefore, in order to confirm that a BTG gene product was produced, western blotting was performed using an anti-BTG antibody prepared in rabbits using purified BTG using Vectastain ABC kit manufactured by Vector. Expression secretion vector pNJ1053-BTG or pNJ1053-pr
o STG-polyacrylamide gel electrophoresis was applied to an amount of about 1 μg in total protein of the cell extract of each yeast holding BTG, and the gel after the electrophoresis was subjected to membrane (Immo).
bilon, Millipore) to bind a protein to be an antigen to the membrane. Thereafter, Western blotting was performed with a rabbit anti-BTG antibody (antibody titer of 64 times diluted 1000 times), and the gene product was confirmed at the protein level.

As a result, a colored band appeared at the same position in the following lanes 1, 3, and 4, and a protein showing a reaction with the anti-BTG antibody could be detected at a position having the same molecular weight as the purified BTG.

1. : Purified BTG (control) : Cell extract of yeast holding pNJ1053 : Cell extract of yeast having pNJ1053-BTG (BTG maturation gene) : Cell extract of yeast holding pNJ1053-proBTG (BTG prosequence-mature gene) The culture supernatant was also subjected to Western blotting for each, but no band was detected in the culture supernatant. Further, in the yeast holding the secretory expression vector pNJ1053-proBTG in which the BTG prosequence-mature gene in lane 4 was incorporated, the processing of the prosequence synthesized in the yeast was performed correctly, so that the position of the same molecular weight as the purified BTG was obtained. It is thought that the band appeared in the event.

[0197]

[Sequence Listing] SEQ ID NO: the length of one sequence: 993 SEQ type: nucleic acid sequence GATTCTGATG ACAGAGTCAC TCCACCAGCT GAACCATTGG ATAGAATGCC AGATCCATAC 60 AGACCATCTT ACGGTAGAGC TGAAACTGTT GTCAACAACT ACATTAGAAA GTGGCAACAA 120 GTCTACTCTC ACAGAGATGG TAGAAAGCAA CAAATGACTG AAGAACAAAG AGAATGGTTG 180 TCTTACGGTT GTGTTGGTGT TACTTGGGTT AACTCTGGTC AATACCCAAC TAACAGATTG 240 GCTTTCGCTT CTTTCGATGA AGATAGATTC AAGAACGAAT TGAAGAACGG TAGACCAAGA 300 TCCGGTGAAA CTAGAGCTGA ATTCGAAGGT AGAGTTGCTA AGGAATCTTT CGATGAAGAA 360 AAGGGTTTCC AAAGAGCTAG AGAAGTTGCT TCTGTTATGA ACAGAGCTCT AGAAAACGCT 420 CACGATGAAT CTGCTTACTT GGATAACTTG AAGAAGGAAT TGGCCAACGG TAACGATGCT 480 TTGAGAAACG AAGATGCTAG ATCCCCATTC TACTCTGCTT TGAGAAACAC TCCATCTTTC 540 AAGGAAAGAA ACGGTGGTAA CCACGATCCA TCCAGAATGA AGGCTGTTAT TTACTCTAAG 600 CACTTCTGGT CTGGTCAAGA TAGATCTTCT TCTGCTGATA AGAGAAAGTA CGGTGATCCA 660 GATGCTTTCA GACCAGCTCC AGGTACCGGT TTGGTCGACA TGTCCAGAGA TAGAAACATT 720 CCAAGATCCC CAACTTCTCC AGGTGAAGGT TTCGTCAACT TCGATTACGG TTGGTTCGGT 780 GCTCAAAC TG AAGCTGATGC TGATAAGACT GTTTGGACCC ATGGTAACCA CTACCACGCT 840 CCAAACGGTT CTTTGGGTGC TATGCACGTC TACGAATCTA AGTTCAGAAA CTGGTCTGAA 900 GGTTACTCTG ATTTCGATAG AGGTGCTTAC GTTATTACTT TCATTCCAAA GTCTGCAGAGCATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCCAGCGTCAGTCAGTGCTGAAGGCTGATCGTAAAA
93 SEQ ID NO: 2 Sequence length: 117 Sequence type: Nucleic acid sequence AAGAGAAGAT CTCCAACTCC AAAGCCAACT GCTTCTAGAA GAATGACTTC TAGACACCAA 60 AGAGCTCAAA GATCTGCTCC AGCTGCTTCT TCTGCTGGTC CATCTTTCAG AGCTCCA 117 SEQ ID NO: 3 Sequence length: TG CGAT CG TG CG CG CGCGACGTGC TTCCGCACGG CCGCGTTCAA 60 CGATGTTCCA CGACAAAGGA GTTGCAGGTT TCCATGCGCT ATACGCCGGA GGCTCTCGTC 120 TTCGCCACTA TGAGTGCGGT TTATGCACCG CCGGATTCAT GCCGTCGGCC GGCGAGGCCG 180 CCGCCGACAA TGGCGCGGGG GAAGAGACGA AGTCCTACGC CGAAACCTAC CGCCTCACGG 240 CGGATGACGT CGCGACATCA ACGCGCTCAA CGAAGCGCTC CGGCCGCTTC GAGCGCCGGC 300 CCGTCGTTCC GGGCCCCCGA CTCCGACGAC AGGGTCACCC CTCCCGCCGA GCCGCTCGAC 360 AGGATGCCCG ACCCGTACCG TCCCTCGTAC GGCAGGGCCG AGACGGTCGT CAACAACTAC 420 ATACGCAAGT GGCAGCAGGT CTACAGCCAC CGCGACGGCA GGAAGCAGCA GATGACCGAG 480 GAGCAACGGG AGTGGCTGTC CTACGGCTGC GTCGGTGTCA CCTGGGTCAA TTCGGGTCAG 540 TACCCCACGA ACAGACTGGC CTTCGCGTCC TTCGACGAGG ACAGGTTCAA GAACGAGCTG 600 AAGAACGGCA GGCCCCGG TC CGGCGAGACG CGGGCGGAGT TCGAGGGCCG CGTCGCGAAG 660 GAGAGCTTTG ATGAAGAGAA GGGGTTCCAG CGGGCGCGTG AGGTGGCGTC CGTGATGAAC 720 AGGGCCCTGG AGAACGCCCA CGACGAGAGC GCTTACCTCG ACAACCTCAA GAAGGAACTG 780 GCGAACGGCA ACGACGCCCT GCGCAACGAG GACGCCCGTT CCCCGTTCTA CTCGGCGCTG 840 CGGAACACGC CGTCCTTTAA GGAGCGGAAC GGAGGCAATC ACGACCCGTC CAGGATGAAG 900 GCCGTCATCT ACTCGAAGCA CTTCTGGAGC GGCCAGGACC GGTCGAGTTC GGCCGACAAG 960 AGGAAGTACG GCGACCCGGA CGCTTTCCGC CCGGCCCCCG GGACCGGCCT GGTCGACATG 1020 TCGAGGGACA GGAACATTCC GCGCAGCCCC ACCAGCCCCG GTGAGGGATT CGTCAATTTC 1080 GACTACGGCT GGTTCGGCGC CCAGACGGAA GCGGACGCCG ACAAGACCGT CTGGACCCAC 1140 GGAAATCACT ATCACGCGCC CAATGGCAGC CTTGGTGCCA TGCATGTATA CGAGAGCAAG 1200 TTCCGCAACT GGTCCGAAGG TTACTCCGAC TTCGACCGCG GAGCCTATGT GATCACCTTC 1260 ATCCCCAAGA GCTGGAACAC CGCCCCCGAC AAGGTAAAGC AGGGCTGGCC GTGATGTGAG 1320 CG 1322

[Brief description of the drawings]

FIG. 1 is a diagram of FIG. Restriction map of clone DNA derived from Streptoverticillium sp.

FIG. 2; B expressed in Streptomyces lividans
Western blot analysis results of TG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 865) (C12N 1/21 C12R 1: 465) (C12N 1/21 C12R 1:19) (C12N 9/10 C12R 1 : 865) (C12N 9/10 C12R 1:19) (C12N 9/10 C12R 1: 465) (C12N 15/09 ZNA C12R 1: 625) (72) Inventor Hiroshi Matsui 1 Suzukicho, Kawasaki-ku, Kawasaki-shi, Kawasaki, Kanagawa Prefecture No. 1 Ajinomoto Co., Inc. Central Research Laboratory (72) Inventor Kinya Washizu 22nd Miyukigaoka Tsukuba, Ibaraki Prefecture Amano Pharmaceutical Co., Ltd.Tsukuba Research Laboratories Co., Ltd. (72) Inventor Keiichi Ando 22nd Miyukigaoka Tsukuba City, Ibaraki Prefecture Amano Pharmaceutical Inside the Tsukuba Research Laboratories Co., Ltd. (72) Inventor Satoshi Koikeda 22nd Miyukigaoka, Tsukuba City, Ibaraki Prefecture Amano Pharmaceutical Co., Ltd. Tsukuba Research Laboratories Co., Ltd. (56) References JP 1-227471 (J , A) JP-T flat 2-503148 (JP, A) (58 ) investigated the field (Int.Cl. ^7, DB name) C12N 15/00 - 15/90 C12N 1/00 - 1/38 C12N 9/00 -9/99 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (17)

(57) [Claims] 1. A DNA comprising a base sequence encoding the amino acid sequence shown below: 2. The DNA according to claim 1, which has the following nucleotide sequence: [Table 2] GAT TCT GAT GAC AGA GTC ACT CCA CCA GCT GAA CCA TTG GAT AGA ATG CCA GAT CCA TAC AGA CCA TCT TAC GGT AGA GCT GAA ACT GTT GTC AAC AAC TAC ATT AGA AAG TGG CAA CAA GTC TAC TCT CAC AGA GAT GGT AGA AAG CAA CAA ATG ACT GAA GAA CAA AGA GAA TGG TTG TCT TAC GGT TGT GTT GGT GTT ACT TGG GTT AAC TCT GGT CAATAC AAC AGA TTG GCT TTC GCT TCT TTC GAT GAA GAT AGA TTC AAG AAC GAA TTG AAG AAC GGT AGA CCA AGA TCC GGT GAA ACT AGA GCT GAA TTC GAA GGT AGA GTT GCT AAG GAA TCT TTC GAT GAA GAA AAG GGT TTC AGA GCT GAA GTT GCT TCT GTT ATG AAC AGA GCT CTA GAA AAC GCT CAC GAT GAA TCT GCT TAC TTG GAT AAC TTG AAG AAG GAA TTG GCC AAC GGT AAC GAT GCT TTG AGA AAC GAA GAT GCT AGA TCC CCA TTC TAC TCT GCT TACT AGA CCA TCT TTC AAG GAA AGA AAC GGT GGT AAC CAC GAT CCA TCC AGA ATG AAG GCT GTT ATT TAC TCT AAG CAC TTC TGG TCT GGT CAA GAT AGA TCT TCT TCT GCT GAT AAG AGA AAG TAC GGT GAT CCA GAT GCT TTC AGA CCA GCT CCA GGT ACC GGT TTG GTC GAC ATG TCC AGA GAT AGA AAC ATT CCA AGA TCC CCA ACT TCT CCA GGT GAA GGT TTC GTC AAC TTC GAT TAC GGT TGG TTC GGT GCT CAA ACT GAA GCT GAT GCT GAT AAG ACT GTT TGG ACC GGT AAC CAC TAC CAC GCT CCA AAC GGT TCT TTG GGT GCT ATG CAC GTC TAC GAA TCT AAG TTC AGA AAC TGG TCT GAA GGT TAC TCT GAT TTC GAT AGA GGT GCT TAC GTT ATT ACT TTC ATT CCA AAG TCT TGG AAC GACT GCT AAG GTC AAG CAA GGT TGG CCA 3. The DNA according to claim 1, further comprising a base sequence encoding all or a part of the amino acid sequence shown below at the 5 ′ end: 4. The DNA according to claim 3, further comprising the following nucleotide sequence at the 5 'end: AAGAGAAGATCTCCAACTCCAAAGCCAACTGCTTCTAGAAGAATGACTTCTAGACACCAA ^{AGAGCTCAAAGATCTGCTCCAGCTGCTTCTTCTGCTGGTCCATCTTTCAGAGCTCCA} 5. The DNA of claim 3 having the nucleotide sequence shown below: TABLE 5 ATGCGCTATA CGCCGGAGGC TCTCGTCTTC GCCACTATGA GTGCGGTTTA TGCACCGCCG GATTCATGCC GTCGGCCGGC GAGGCCGCCG CCGACAATGG CGCGGGGGAA GAGACGAAGT CCTACGCCGA AACCTACCGC CTCACGGCGG ATGACGTCGC GACATCAACG CGCTCAACGA AGCGCTCCGG CCGCTTCGAG CGCCGGCCCG TCGTTCCGGG CCCCCGACTC CGACGACAGG GTCACCCCTC CCGCCGAGCC GCTCGACAGG ATGCCCGACC CGTACCGTCC CTCGTACGGC AGGGCCGAGA CGGTCGTCAA CAACTACATA CGCAAGTGGC AGCAGGTCTA CAGCCACCGC GACGGCAGGA AGCAGCAGAT GACCGAGGAG CAACGGGAGT GGCTGTCCTA CGGCTGCGTC GGTGTCACCT GGGTCAATTC GGGTCAGTAC CCCACGAACA GACTGGCCTT CGCGTCCTTC GACGAGGACA GGTTCAAGAA CGAGCTGAAG AACGGCAGGC CCCGGTCCGG CGAGACGCGG GCGGAGTTCG AGGGCCGCGT CGCGAAGGAG AGCTTTGATG AAGAGAAGGG GTTCCAGCGG GCGCGTGAGG TGGCGTCCGT GATGAACAGG GCCCTGGAGA ACGCCCACGA CGAGAGCGCT TACCTCGACA ACCTCAAGAA GGAACTGGCG AACGGCAACG ACGCCCTGCG CAACGAGGAC GCCCGTTCCC CGTTCTACTC GGCGCTGCGG AACACGCCGT CCTTTAAGGA GCGGAACGGA GGCAATCACG ACCCGTCCAG GATGAAGGCC GTCA TCTACT CGAAGCACTT CTGGAGCGGC CAGGACCGGT CGAGTTCGGC CGACAAGAGG AAGTACGGCG ACCCGGACGC TTTCCGCCCG GCCCCCGGGA CCGGCCTGGT CGACATGTCG AGGGACAGGA ACATTCCGCG CAGCCCCACC AGCCCCGGTG AGGGATTCGT CAATTTCGAC TACGGCTGGT TCGGCGCCCA GACGGAAGCG GACGCCGACA AGACCGTCTG GACCCACGGA AATCACTATC ACGCGCCCAA TGGCAGCCTT GGTGCCATGC ATGTATACGA GAGCAAGTTC CGCAACTGGT CCGAAGGTTA CTCCGACTTC GACCGCGGAG CCTATGTGAT CACCTTCATC CCCAAGAGCT GGAACACCGC CCCCGACAAG GTAAAGCAGG GCTGGCCG 6. The DNA according to claim 1, which is obtained by chemical synthesis. 7. The DNA according to claim 1, 3 or 5, which is obtained by cloning. 8. The DN according to any one of claims 1 to 7,
An expression secretion vector incorporating A. 9. The expression secretion vector according to claim 8, which is pOMPA-BTG. 10. The expression secretion vector according to claim 8, which is pNJ1053-proBTG. 11. The expression secretion vector according to claim 8, which is pNJ1053-BTG. 12. The expression secretion vector according to claim 8, which is pIJ702-BTG. A transformant transformed by the expression secretion vector according to any one of claims 8 to 12. 14. The transformant according to claim 13, which is Escherichia coli. 15. The method according to claim 1, wherein the yeast is yeast.
3. The transformant according to 3. 16. The transformant according to claim 13, which is an actinomycete. 17. A method for producing a protein having transglutaminase activity, which comprises culturing the transformant according to claim 13.