JPH04335883A - Production of antibacterial polypeptide - Google Patents

Abstract

PURPOSE:To abundantly produce the subject polypeptide showing a wide anti-bacterial spectrum at a low cost by culturing a yeast transformant in which a recombinant plasmid containing DNA capable of coding an antibacterial polypeptide sapecin is integrated. CONSTITUTION:A Sacrophaga peragrina Robineau-Desvoidy embryo-derived established cell line is cultured and a genom gene is extracted from the resultant cells by the conventional method. A gane corresponding to mutation protein part is amplified by PCR method, then treated with a restriction enzyme and subsequently subjected to agarose electrophoresis to recover, using a probe, a gane for Sapecin which is an anti-bacterial polypeptide having an amino acid sequence represented by the formula. To the resultant gene, a base sequence of a signal peptide required for secretory manifestation and a base sequence required for completion of transcription are bonded so that secretory magnifestation may be possible in a yiest. A recombinant DNA is obtained thereby and the obtained DNA is then induced into a microorganism such as a yiest to transform it. The resultant transformant is then cultured, thus giving the objective Specin as an antibacterial polypeptide containing an amino acid sequence represented by the formula.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a transformation that incorporates a gene encoding an antibacterial polypeptide that is induced into the body fluid of insect larvae belonging to the order Diptera or Hymenoptera when the body surface of the larva is damaged. The present invention relates to a method for producing the antibacterial polypeptide by culturing the transformant.

More specifically, a polypeptide with antibacterial properties, ie, zapecin, is induced into body fluids by wounding the body surface of the larva of the centinic fly.
The present invention relates to a method for producing zerpecin by incorporating a gene encoding a host microorganism into yeast, transforming the yeast, and culturing the yeast.

0003

BACKGROUND OF THE INVENTION It is known that a number of proteins are induced in body fluids when a stimulus is applied, such as inoculating an insect with bacteria or blood cells of a different species, or simply scratching the insect's body surface. These substances are considered to have an important role in the biological defense of animals that do not have the ability to produce antibodies.

Among these, for example, the centinic fly (
Sarcophaga peregrina) A protein with a hemagglutinating effect is an active protein induced in the body fluid of larvae [J. Biol. Chem. , 255 volumes,
2919-2924 (1980)], proteins with antibacterial activity [Biochem. J. , vol. 211, 727-
734 pages (1983)] and others have been identified.

The former lectin-like protein is named Sarcophaga lectin, and it enhances the fungicidal ability of mouse bone marrow cells and macrophages.
induces the production of γ-interferon from human T cells,
Effects such as biological defense are being studied.

[0006] As for the latter protein with antibacterial activity,
For example, a protein has been named Sarcotoxin I and its physicochemical properties have been clarified (Japanese Patent Laid-Open No. 59-13730), and a protein has been named Sarcotoxin II and its physicochemical properties have been clarified (JP 59-13730). 63-35599) protein, also obtained from the larval hemolymph of the centinic fly, Sapecin
There is a known protein which has been isolated and whose amino acid sequence has been determined (Japanese Patent Application Laid-Open No. 185997/1983).

[0007] Since these have a broad antibacterial spectrum, they are considered to be biological defense substances, and furthermore, these antibacterial proteins have particularly strong antibacterial activity against Escherichia coli, a gram-negative bacterium, and Staphylococcus aureus, a gram-positive bacterium. are doing. However, there has been no report on a method for producing these proteins in large quantities at low cost.

Regarding sarcotoxin, cloned DNA of its precursor, its fragments, and plasmids incorporating them have been reported (Japanese Patent Application Laid-Open Nos. 1-51084 and 1-51085). However, the gene encoding the protein for zerpecin has not been obtained. Of course, there has been no report on a method for producing the protein by incorporating the gene into a host microorganism.

[0009]

[Problems to be Solved by the Invention] The present invention provides antibacterial proteins,
In particular, the present invention provides a plasmid that can be expressed using yeast as a host and has a DNA encoding zerpecin integrated therein, a recombinant in which the plasmid is integrated into yeast, and a method for producing zerpecin by culturing the recombinant.

Since zerpecin has antibacterial activity, it cannot be produced by conventional genetic recombination methods using Escherichia coli. The present invention makes it possible for the first time to produce a large amount of the antibacterial protein zerpecin by selecting yeast as a recombinant host.

[0011]

[Means for Solving the Problems] As a result of intensive research, the present inventors constructed a gene for zerpecin, obtained a transformed yeast incorporating the gene, and produced zerpecin by culturing the transformant. They were very successful and completed the present invention.

That is, according to the present invention, zerpecin shown in SEQ ID NO: 1 can be produced in large quantities at low cost, and the obtained protein can be used as an antibacterial agent. Hereinafter, the present invention will be explained in detail with reference to Examples and Test Examples.

[0013]

【Example】

Example 1 Construction of Zarpecin gene■ Preparation of Zarpecin gene fragment The Zarpecin gene was obtained by culturing NIH-Sape-4 cells, which are established cell lines derived from centinic fly embryos.
l. Chem. ), vol. 263, 17117-1712
1 (1988)]. The mature protein portion (shown in SEQ ID NO: 1) of the gene was amplified by PCR. Primer 1 (shown in SEQ ID NO: 2) and primer 2 (shown in SEQ ID NO: 3) were used as primers.

[0014] Primer 1 contains part of the base sequence (including the HindIII site) of the signal peptide of α factor. Primer 2 also contains a stop codon and an EcoRI site.

[0015] The reaction of the PCR method was performed using Gene AmpTM.
DNA using kit [manufactured by PerkinElmer Japan]
This was carried out using a Thermal Cycler (manufactured by PerkinElmer Japan). The composition of the reaction solution was determined according to the method described in the instructions attached to the kit.

[0016] Transfer the tube containing the reaction solution to DNA Th.
The reaction mixture was set in an ermal cycler, and the reaction was carried out for 35 cycles under the following conditions for amplification. 95℃, 1 minute 40℃, 2 minutes 72℃, 3 minutes

[0017] Thereafter, the mixture was further incubated at 72°C for 7 minutes. After the reaction, the reaction solution was taken out and extracted with chloroform. Next, this was electrophoresed on a 1.5% agarose gel to confirm the size and amount of the amplified DNA. As a result, approximately 0.2 μg of approximately 150 bp DNA fragment
Obtained.

[0018] Take out the gel containing this 150 bp band, and use MERMAID Kit (manufactured by Bio 101) to perform DNA analysis according to the instructions attached to the kit.
A was extracted and collected. Furthermore, the recovered DNA was digested with restriction enzymes EcoRI (manufactured by Toyobo Co., Ltd.) and HindIII (manufactured by Toyobo Co., Ltd.), and subjected to agarose electrophoresis.
MERMAID 50bp zerpecin gene fragment
It was collected using Kit.

■ Addition of a yeast secretion signal and a transcription terminator to the zarpecin gene In order to express the obtained zarpecin gene fragment, the base sequence of the signal peptide necessary for secretory expression and transcription are added to enable secretory expression in yeast. The nucleotide sequence (terminator) required for termination of the terminator was ligated to the zerpecin gene fragment. The process is shown in Figure 1.
and shown in FIG.

More specifically, first, the base sequence of the signal peptide portion of yeast α-factor was amplified and isolated using the above-mentioned PCR method. In other words, yeast genomic DNA
(purchased from Clontec) was used as a template DNA, and the base sequence of the signal peptide portion of α factor was amplified using primer 3 (shown in SEQ ID NO: 4) and primer 4 (shown in SEQ ID NO: 5). After the amplification, the reaction solution was subjected to agarose electrophoresis as described above to confirm the amplified DNA fragments, which were extracted and recovered using the MERMAID Kit. As a result, about 0.5 μg of about 300 bp D
An NA fragment was obtained.

[0021] Here, primer 3 is a restriction enzyme XhoI
Since primer 4 contains a site for the restriction enzyme HindIII, the resulting DNA fragment was digested with XhoI and HindIII to create a DNA fragment containing the base sequence of the α-factor signal peptide at both ends. XhoI and HindIII sites can be introduced respectively.

[0022] The DNA fragment containing the base sequence of the α-factor signal peptide obtained above was digested with XhoI and HindI.
II (both manufactured by Toyobo Co., Ltd.) to digest Blue Script ks- (manufactured by Stratagene) with XhoI and Hi
It was ligated with the large fragment digested with ndIII. The ligation reaction was carried out using a ligation kit (manufactured by Takara Shuzo Co., Ltd.) according to the instructions attached to the kit. After the ligation, Escherichia coli DH5 (manufactured by Toyobo Co., Ltd.) was transformed with the reaction solution. Transformation can be carried out using known methods such as [Molecular Cloning (Mol.
(1989)].

As a result of the transformation, plasmid DNA was extracted from the ampicillin-resistant strain obtained by known methods such as [
Molecular cloning
Cloning (1989)]. This plasmid was named pBα.

Next, this plasmid pBα was transformed into HindII
The large fragment obtained by cutting with I and EcoRI (both manufactured by Toyobo Co., Ltd.) and the aforementioned zarpecin gene were
The fragments cut with ndIII and EcoRI were ligated.

After ligation, Escherichia coli DH5 was transformed using this in the same manner as described above. A plasmid (hereinafter referred to as plasmid pBSα) was prepared from the obtained transformant as described above and used in the following experiments.

[0026] Next, the terminator sequence of the yeast PGK gene was inserted downstream of the zarpecin gene in the plasmid pBSα.

First, the terminator sequence of the PGK gene was amplified and isolated from yeast genomic DNA. That is, mold D
It was amplified by PCR using yeast genomic DNA as NA and primers 5 (shown in SEQ ID NO: 6) and primer 6 (shown in SEQ ID NO: 7) as primers. The reaction solution was subjected to agarose electrophoresis, and the amplified DN
The A fragment was confirmed, extracted and recovered using MERMAID Kit. As a result, approximately 0.5 μg of approximately 30
A 0 bp DNA fragment was obtained.

Primer 6 contains an EcoRI site, and primer 7 contains a BamHI site. By digesting the obtained fragment with EcoRI and BamHI (manufactured by Toyobo Co., Ltd.), EcoRI sites were created at both ends of the terminator sequence of the PGK gene. and the BamHI site can be introduced.

The obtained terminator fragment of the PGK gene was digested with EcoRI and BamHI, and this and the large fragment obtained by digesting the plasmid pBSα with EcoRI and BamHI were ligated in the same manner as described above, and transformation was performed.

Plasmid DNA was prepared from the ampicillin-resistant strain obtained and named plasmid pBGSα, and used in the following experiments.

The base sequence of the obtained plasmid pBGSα was investigated using a sequence kit (manufactured by Toyobo Co., Ltd.). As a result, it was confirmed at the base sequence level that the nucleotide sequence of the alpha factor signal peptide is bound upstream of the zerpecin gene, and the terminator sequence of the PGK gene is bound downstream.

[0032] Furthermore, when we investigated in more detail the base sequence of the signal peptide portion of α factor and the base sequence of the upstream link of the zarpecin gene, we found that the base sequence was as designed, and is shown in SEQ ID NO: 8. It was something like that.

When this part is sequentially translated into amino acids starting from methionine, which is the start codon of α factor, it becomes glycine-valine-serine-leucine-aspartic acid-lysine-arginine-alanine-threonine-cysteine-aspartic acid-leucine-leucine. This becomes an array.

Among these, the amino acid sequence from the first glycine to the seventh arginine is the amino acid sequence of α factor, and from the eighth alanine to the amino acid sequence of the N-terminal region of the mature form of zerpecin. The lysine-arginine sequence in this sequence is the part where the α-factor signal peptide is processed, and it is predicted that the mature protein of zerpecin is excised and secreted at this part.

Example 2 Cloning of Zerpecin Gene Plasmid pBGSα was digested with XhoI and BamHI, subjected to agarose electrophoresis, a DNA fragment of approximately 750 bp was identified, and this fragment was recovered using a MERMAID kit. This fragment contains the zarpecin gene, which has the above-mentioned alpha factor signal peptide nucleotide sequence upstream and the PGK terminator downstream. This DNA fragment was transferred to yeast
It was cloned into pAM82B, an E. coli shuttle vector.

Vector pAM82B is a derivative of pAM82, and contains Ba at the PvuII site of pAM82.
A BamHI site was introduced by incorporating an mHI linker.

[0037] Immediately upstream of the XhoI site of pAM82B, the promoter of the yeast acid-inhibitory phosphatase gene PHO5 is placed, and by inserting a gene with an appropriate structure downstream of the XhoI site, the gene can be expressed. can.

That is, pAM82B was mixed with XhoI and BamH.
The large fragment digested with I and the aforementioned 750 bp
This DNA fragment was ligated to E. coli DH5 using this DNA fragment.

Approximately 500 μg of a plasmid (hereinafter referred to as plasmid pAM82B-Sap) was prepared from the obtained ampicillin-resistant strain and used in the following experiment.

Example 3 Plasmid pAM82B-S
Transformation of yeast SHY2 by ap Yeast SHY2 (ATCC No. 44770) was transformed using the plasmid pAM82B-Sap obtained above. The method of transformation is [Laboratory
Course Manual for Me
thods In Yeast Genetic
s(Cold Spring Harbor L
Laboratory)].

[0041] The properties of yeast SHY2 are (α, ste-VC
9, ura3-52, trp1-289, leu-3,
leu2-112, his3-1, can1-100)
This strain cannot grow on a medium without leucine. If this strain retains plasmid pAM82B, it will become a strain that can grow even in a medium that does not contain leucine due to the action of the leucine synthesis gene contained in plasmid pAM82B, that is, it will become a leucine non-auxotrophic strain. Therefore, the yeast transformed by the plasmid pAM82B-Sap becomes leucine non-auxotrophic.

As a result of the transformation, five transformants were obtained. Among them, Saccharomyces cerevisiae (Sac
charomyces cerevisiae)YS
A transformant named YS2 (hereinafter referred to as YS2) [This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology for the 12th time.
No. 237 (FERM P-12237). ] was used in the following experiment.

Example 4 Culture of YS2 Obtained YS2
The culture was carried out as follows. YS2 with high phosphate Bu
The cells were inoculated into 10 ml of rkholder minimal medium and precultured overnight at 30°C. Furthermore, Burkhold
The composition of the er minimal medium is as shown below. (NH4)2SO4
2
g/l KH2PO4

1.5 g/l MgSO4・7H2
O
0.5 g/l CaCl2・2
H2O
0.33 g/l KI

0.1 mg/l
CuSO4・5H2O
0.04 mg/l
FeSO4・7H2O
0.25 mg/l
MgSO4・4H2O
0.04 mg/l
(NH4)3PO412MoO3・3H2O
0.02 mg/l ZnSO4・
7H2O
0.31 mg/l inositol
10 mg/l thiamine
0.2 mg/l
pyridoxine
0.2 mg
/l pantotenate (Ca sal
t) 0.2 mg/l niacin
0.2 mg/l
biotin
0.002mg/l
asparagine
2
g/l histidine
0.
02 mg/l tryptophan
0
．． 025mg/l uracil

0.020mg/l glucose

20g/l

[0044] Transfer 0.5 ml of the preculture solution to 100 ml of high phosphate Burkhol.
The cells were inoculated into a derminimal medium, cultured with shaking at 30°C, and OD610 was measured over time.

[0045] When OD610 reached 1.0, the culture was temporarily stopped and the bacterial cells were collected by centrifugation (8000 x g, 5 minutes). The collected bacterial cells were then added to 100 ml of low phosphate Bu.
rkholder minimal medium (high phosphate B
KH2 from urkholder minimal medium
After removing PO4, the cells were suspended in 1.5 g/l of KCl and cultured with shaking for 24 hours.

After culturing, the bacterial cells were centrifuged (8000×g,
5 minutes), and the culture supernatant was collected and used in the following experiment. As a control, plasmid pA
A culture supernatant recovered by performing the same operation as above using SHY2 (hereinafter referred to as YS0) transformed with M82B was used.

Example 5 Concentration of Culture Supernatants In order to examine whether the above-mentioned culture supernatants contained the expressed zerpecin, the culture supernatants of YS2 and YS0 were each concentrated.

A CM cellulose column (Whatman CM52; bed volume 5 ml) was used to concentrate the culture supernatant. First, add 100 ml of culture supernatant to buffer A.
It was diluted 5 times. Next, add 5 CM cellulose columns.
After washing with 0 ml of buffer B, this diluted culture supernatant was applied to the column. After applying, add 5 more columns.
After washing with 0 ml buffer B, 500 ml buffer C
The components adsorbed on the column were eluted and collected in 0.5 ml portions using a fraction collector. The OD280 of each fraction was measured, and these were used as CM cellulose adsorption fractions in the following experiment. The compositions of buffer solution A, buffer solution B, and buffer solution C are as shown below.

■ Buffer A NaH2PO4 1.37g/
lNa2HPO4 0.44g
/l■ Buffer B NaH2PO4 1.37g/
lNa2HPO4 0.44g
/lNaCl 1.4
6g/l■ Buffer C NaH2PO4 1.37g/
lNa2HPO4 0.44g
/lNaCl 30.
4g/l

Test Example 1 Measurement of antibacterial activity of CM cellulose adsorbed fraction The antibacterial activity of each CM cellulose adsorbed fraction of the culture supernatants of YS2 and YS0 was measured as follows.

Antibiotic medium
Staphylococcus aureus (St.
aphylococcus aureus)FDA2
09P strain was inoculated into 10 ml of M3 liquid medium and incubated at 37°C under O
Shaking culture was performed until D650=0.3.

After culturing, the bacterial cells were collected by centrifugation (8000×g, 15 minutes) and added to buffer D [30mM potassium phosphate buffer (pH 7.0), 60mM NaCl].
] was suspended so that the OD650 was exactly 0.3. 0.04 ml of this suspension, 0.16 ml of M3 liquid medium, 0.1 ml of each CM cellulose adsorption fraction, and 0.2% BS.
0.1 ml of buffer D containing A was mixed in a test tube and cultured with shaking at 37°C for 3 hours. After culturing, the test tube was cooled in ice for 1 hour, and then the OD650 of the culture solution was measured. The OD650 value of the culture solution when 0.1 ml of buffer C was added instead of the CM adsorption fraction was used as a control.
Staphylococcus aureus F by the following formula
The growth rate of the DA209P strain was calculated. Proliferation rate = (OD650 in each fraction/OD65 of control
0)×100

Figure 3 shows the proliferation rate calculated for each fraction and the OD280 value of each fraction.
and shown in FIG.

[0054] In the culture supernatant of YS0, the peak of OD280 was observed in fractions 6 to 11. At this time, the proliferation rate for each fraction was all 100 or more, and no antibacterial activity was observed for any fraction.

On the other hand, in the culture supernatant of YS2, peaks of OD280 were observed in fractions 2-5 and 9-12. At this time, it was found that the proliferation rate for each fraction was significantly reduced in fractions 10 and 11. Furthermore, the culture supernatant obtained when fractions 9 to 13 were added was spread on an M3 plate, cultured overnight at 37°C, and the number of formed colonies was measured. was significantly less. Therefore, the reason why the OD650 was low in the culture solution containing fractions 10 and 11 is considered to be because the number of viable bacteria in the culture solution was small. Therefore, it was revealed that fractions 10 and 11 had antibacterial activity.

From the above, it was revealed that the culture supernatant of YS2 contains a substance with antibacterial activity. Since this substance with antibacterial activity was not present in the culture supernatant of YS0, it is clear that its expression depends on the presence of the zerpecin gene. Therefore, the substance with this antibacterial activity is considered to be zerpecin expressed in yeast.

Test Example 2 Western blot analysis of CM cellulose adsorbed fraction In order to further confirm that the substance exhibiting antibacterial activity in fractions 10 and 11, which are CM cellulose adsorbed fractions of the culture supernatant of YS2, was zerpecin. Western blot analysis was performed using these two fractions.

[0058] The Western blot analysis method is generally based on molecular cloning (Molecular C
(1989).

[0059] Each sample shown below was subjected to SDS polyacrylamide electrophoresis, and the gel was stained with Coomassie blue. The results are shown in Figure 5. The same electrophoresis was also performed, and Western blot analysis was performed using rabbit anti-Zerpecin antibody. The results are shown in FIG. The following samples are applied to each lane. Lane 1; Purified serpecin Lane 2; YS2 culture supernatant CM cellulose adsorption fraction,
Fraction 10 Lane 3; Same as above, Fraction 11 Lane 4; YS0 culture supernatant CM cellulose adsorption fraction, Fraction 11 Lane 5; Same as above, Fraction 12

First, in the results of Coomassie blue staining, a single band was observed in lane 1. This is Zarpecin. In lanes 2 and 3 to which the CM cellulose adsorbed fraction of the culture supernatant of YS2 was applied, one band was observed at the same position, and no other bands were observed.

On the other hand, no bands were observed in lanes 4 and 5 to which the CM cellulose adsorbed fraction of the culture supernatant of YS0 was applied.

Next, according to the results of Western blot analysis,
In lane 1 to which Zerpecin was applied, one signal was observed at the position of the band observed by Coomassie blue staining. At this time, signals were observed at the same position in lanes 2 and 3 with approximately the same intensity. Also, no signal was observed in lanes 4 and 5.

From the above, it was found that a substance having the same antigenicity and the same molecular weight as zerpecin was expressed in the culture supernatant of YS2. The expression of this substance was dependent on the presence of the zarpecin gene, and it was concluded that this was zerpecin expressed in yeast.

[0064] By the way, the bands observed in lanes 2 and 3 in the Coomassie blue staining results are considered to be caused only by zerpecin since they are not observed in lanes 4 and 5.

[0065] Since no bands due to proteins other than zerpecin were observed, it is considered that the product has already been purified considerably at this point. That is, according to this method, it is considered that zerpecin can be purified quite easily. Furthermore, the amount of zerpecin produced in YS2 was estimated to be about 0.5 mg/l based on the results of Western blot analysis.

This value is comparable to the expression level of zerpecin in the insect-derived cell NIH-sape-4 which naturally produces zerpecin.

[0067]

[Effects of the Invention] Since yeast culture is more economical and technically simpler than insect cell culture, YS2 is an excellent material for preparing zerpecin. Moreover, as mentioned above, it is expected that the purification will be simple. From the above, using this method, zerpecin can be prepared in large quantities and at low cost.

[Sequence list]

SEQ ID NO: 1 Sequence length: 40 Sequence type: Amino acid Topology: Linear Sequence type: Genomic DNA Sequence Ala Tyr Cys Asp Leu Leu S
er Gly Thr Gly Ile Asn Hi
s Ser Ala 15Cys A
la Ala His Cys Leu Leu Ar
g Gly Asn Arg Gly Gly Tyr
Cys 30Asn Gly Ly
s Ala Val Cys Val Cys Arg
Asn
40

SEQ ID NO: 2 Sequence length: 40 Sequence type: Nucleic acid Topology: Linear Sequence type: Fragment sequence GGTAAGCTTG GATAAAAGAG C
CACTTGCGA 30TTTATTGAGT

40

SEQ ID NO: 3 Sequence length: 32 Sequence type: Nucleic acid Topology: Linear Sequence type: Fragment sequence CGGAATTCTT AATTGCGACA T
ACGCAGACA 30GC


32

SEQ ID NO: 4 Sequence length: 34 Sequence type: Nucleic acid Topology: Linear Sequence type: Fragment sequence GCCTCGAGTT TCATACACAAT
ATAAACGAC 30CAAA


34

SEQ ID NO: 5 Sequence length: 34 Sequence type: Nucleic acid Topology: Linear Sequence type: Fragment sequence GGAAGCTTAC CCCTTCTTCT T
TAGCAGCAA 30TGCT


34

SEQ ID NO: 6 Sequence length: 33 Sequence type: Nucleic acid Topology: Linear Sequence type: Fragment sequence CCGAATTCAT TGAATTGAAT T
GAAATCGAT 30AGA


33

SEQ ID NO: 7 Sequence length: 41 Sequence type: Nucleic acid Topology: Linear Sequence type: Fragment sequence CCGGATCCGA TATCGGTTTT T
CGAAACGCA 30GAATTTTTCGA

40

SEQ ID NO: 8 Sequence length: 39 Sequence type: Nucleic acid Topology: Linear Sequence type: Fragment sequence GGGGTAAGCT TGGATAAAAAG A
GCCACTTGC 30GATTTATTG

3
9

[Brief explanation of drawings]

FIG. 1 shows the steps for creating plasmid pBGSα.

[Figure 2] From plasmid pBGSα to plasmid pAM8
The process of creating 2B-Sap is shown.

FIG. 3 shows the proliferation rate calculated for each fraction of the culture supernatant of YS2 and the OD280 value of each fraction, where -●- indicates the proliferation rate, and -○- indicates the OD280.
indicates the value of

FIG. 4 shows the proliferation rate calculated for each fraction of the culture supernatant of YS0 and the OD280 value of each fraction, where -●- indicates the proliferation rate, and -○- indicates the OD280.
indicates the value of

FIG. 5 shows the results of SDS polyacrylamide electrophoresis performed on purified zerpecin, CM cellulose adsorbed fractions of YS2 and YS0 culture supernatants, and the gel stained with Coomassie blue.

FIG. 6 shows the results of SDS polyacrylamide electrophoresis and Western blot analysis using a rabbit anti-Zerpecin antibody for each fraction, which is the CM cellulose-adsorbed fraction of purified Zerpecin, YS2 and YS0 culture supernatants.

Claims (2)

[Claims] Claim 1: A nucleotide sequence encoding an antibacterial polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 is ligated to a replicable expression vector to produce a clone containing the nucleotide sequence and the replicable expression vector. Possible recombinant DNA
A transformant obtained by transforming a microorganism with the recombinant DNA. 2. A nucleotide sequence encoding an antibacterial polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 is ligated to a replicable expression vector to produce a clone containing the nucleotide sequence and the replicable expression vector. Possible recombinant DNA
obtained, transform a microorganism with the recombinant DNA to obtain a transformant, and culture the transformant.
A method for producing an antibacterial polypeptide containing the amino acid sequence shown in 1.