JPH10117783A - Immobilization of protein on surface of microbial cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To express various useful proteins by immobilizing the various useful proteins on the surface of a microbial cell by utilizing an anchor sequence of a protease derived from Lactococcus lactis. SOLUTION: A fused gene sequence obtained by combining a gene sequence coding an anchor sequence (or a partial sequence thereof) of 763 protease derived from Lactococcus lactis NCD 0763 strain, with a gene sequence coding the objective protein is introduced into a host microbe. The objective protein is immobilized on the surface of the host microbial cell by culturing the host microbe. The anchor sequence having the amino acid sequence of the formula is preferable. An application to an oral vaccine is expected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a lactococcus
The present invention relates to an immobilization method for expressing any useful protein on the surface of a host cell using an anchor sequence of a protease protein on the surface of a cell of lactis ( Lactococcus lactis ).

[0002]

2. Description of the Related Art Many cell surface proteins of gram-positive bacteria have a common structure called an anchor sequence, and are bound to the cells via the structure. Among the proteases of Lactococcus lactis, the so-called 763 protease is
Lactococcus lactis ssp. Lactis is a serine protease whose production information is retained on the 55Kb plasmid (pLP763) of the NCDO763 strain, which takes on a structure that binds to the cells on the surface of the cells by means of an anchor sequence located at the C-terminus and is expressed. It is known.

[0003] This anchor sequence is a structure possessed by many cell surface proteins of Gram-positive bacteria, (1) a proline- and glycine-rich repeat region located in a peptidoglycan layer constituting a cell wall, and (2) an amino acid residue. Consensus sequence consisting of groups (LPXTG motif),
It consists of (3) a hydrophobic region located in the cell membrane, and (4) a positively charged region thought to be located in the cytoplasm.

Regarding the anchor sequence of 763 protease, its amino acid structure has been reported (see literature; Molecular Microbiology (1989) vol. 3 (No. 3), 359-).
369) and 763 protease, the proline / glycine-rich repeat region described in (1) above is not clear, and whether all of them are essential or not essential for protease binding to bacterial cells. In such a case, it is not sufficiently clarified which part is the minimum necessary unit.

[0005]

The expression of a useful protein on the surface of bacterial cells can be an effective means for breeding microorganisms. Thus, as a result of diligent efforts, the present inventors have elucidated a region involved in binding of a protease to bacterial cells in an anchor sequence derived from the 763 protease, and thereby obtained the present invention.

According to the present invention, various useful proteins are immobilized on the surface of bacterial cells using the anchor sequence of a protease derived from Lactococcus lactis or a partial sequence of the anchor sequence, and the useful protein is expressed. The purpose is to let them.

[0007]

According to the method for immobilizing a protein on the surface of a bacterial cell according to the present invention described in claim 1,
Lactococcus lactis NCDO7
A gene sequence encoding the anchor sequence of 763 protease from the 63 strain and a step of obtaining a fusion gene sequence connecting the gene sequence encoding the protein of interest, and a step of introducing the fusion gene sequence into a host bacterium. Culturing a host cell into which the fusion gene sequence has been introduced, and immobilizing the target protein on the surface of the cell body.

[0008] In the method for immobilizing a protein on the surface of a bacterial cell according to the invention described in claim 2, the anchor sequence has at least the following amino acid sequence. Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln Arg Glu Glu

In the method for immobilizing a protein on the surface of a bacterial cell according to the present invention described in claim 3, the anchor sequence is obtained by removing a part of the following amino acid sequence except for the amino acid sequence shown in claim 2. It consists of an amino acid sequence. Lys Lys Thr Ser Leu Leu Asn Gln Leu Gln Ser Val Lys Ala Ala Leu Glu Thr Asp Leu Gly Asn Gln Thr Asp Ser Ser Thr Gly Lys Thr Phe Thr Ala Ala Leu Asp Asp Leu Val Ala Gln Ala Gln Ala Gly Thr Gln Thr Asp Asp Gln Leu Gln Ala Thr Leu Ala Lys Val Leu Asp Ala Val Leu Ala Lys Leu Ala Glu Gly Ile Lys Ala Ala Thr Pro Ala Glu Val Gly Asn Ala Lys Asp Ala Ala Thr Gly Lys Thr Trp Tyr Ala Asp Ile Ala Asp Thr Leu Thr Ser Gly Gln Ala Ser Ala Asp Ala Ser Asp Lys Leu Ala His Leu Gln Ala Leu Gln Ser Leu Lys Thr Lys Val Ala Ala Ala Val Glu Ala Ala Lys Thr Val Gly Lys Gly Asp Gly Thr Thr Gly Thr Ser Asp Lys Gly Gly Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly Asp Ile Gly Lys Asp Lys Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly Gly Asn Ile Pro Thr Asn Pro Ala Thr Thr Thr Ser Ser Serr Thr Asp Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln Arg Glu Glu

[0010] The target protein in the present invention refers to a useful protein expressed on the surface of a lactic acid bacterium as a host according to the culture of the lactic acid bacterium. For example, enzymes such as staphylokinase (SAK) shown in Examples described later, partial proteins of pathogenic bacteria, bioactive proteins, and the like are also considered. That is, there is no particular limitation as long as the gene of any protein is isolated and can be combined with the anchor sequence using a normal gene recombination technique to produce the gene of the fusion protein.

[0011] The host strain of the present invention, the anchor sequence of Lactococcus lactis (Lactococcus l actis) NCDO763 strain derived from 763 protease takes a structure which binds to cells in the cell surface, the cell surface Any expression can be used. Specifically, Lactococcus lactis , Lactobacillus casei
(Lactobacillus casei), Streptococcus thermophilus (Streptococcus thermophilus), Enterococcus faecalis (Enterococcus faecalis), Staphylococcus aureus (Staphylococcus aureus), Streptococcus mutans (Streptococcus mutan
s), Streptococcus pyogenes (Streptococcus p
lactic acid bacteria such as yogenes ).

A gene sequence encoding an anchor sequence;
Preparation of a fusion gene sequence linked to the gene sequence encoding the protein of interest, introduction of the fusion gene sequence into a host cell, and cultivation of the host cell into which the fusion gene sequence has been introduced must be carried out by a conventional method. Can be.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a fusion gene comprising a gene sequence encoding an anchor sequence of Lactococcus lactis NCDO763 strain-derived 763 protease and a gene sequence encoding a target protein is described. After obtaining the sequence, the fusion gene sequence is introduced into a host bacterium, and this is cultured to immobilize the target protein on the surface of the cell.

[0014] Thus, for example, the enzyme can be expressed and immobilized on the surface of the host cell, a vaccine can be obtained by using a partial protein of a pathogenic bacterium, or a bioactive protein can be obtained by using a bioactive protein. It can be used for pharmaceutical purposes. In the examples described below, staphylokinase (SA) having an excellent thrombolytic action is described.
K) Although the protein was used as a model, various changes can be made according to the target protein.

Introduction of a gene sequence encoding a fusion protein of an anchor sequence and a target protein into a host bacterium can be carried out using a conventional gene recombination technique. For example, it can be carried out by retaining the gene sequence in a plasmid that can be introduced into a lactic acid bacterium serving as a host bacterium, and introducing this plasmid into the host bacterium.

[0016] Lactococcus lactis (Lactococcus l
actis ) For the anchor sequence of the 763 protease derived from the NCDO763 strain, it was confirmed for the first time by the present invention that the 243 amino acid residue sequence to 36 amino acid residue sequence shown in SEQ ID NOs: 1 to 6 can be used. In particular, in the present invention, the 36 amino acid residue sequence shown in SEQ ID NO: 6 contains Lactococcus
It has been confirmed that it can be used in Lactis and Lactobacillus casei.

Specifically, (1) 243 amino acid residue sequence shown in SEQ ID NO: 1 (2) 158 amino acid residue sequence shown in SEQ ID NO: 2 (3) 125 amino acid residue sequence shown in SEQ ID NO: 3 Residue sequence (4) 96 amino acid residue sequence shown in SEQ ID NO: 4 (5) 55 amino acid residue sequence shown in SEQ ID NO: 5 (6) 36 amino acid residue sequence shown in SEQ ID NO: 6 It has been confirmed that this is possible, and other subsequences can be used within these ranges.

More specifically, the sequence shown in SEQ ID NO: 2 has a structure in which 85 amino acid residues on the N-terminal side of SEQ ID NO: 1 have been removed. The sequence shown in SEQ ID NO: 3 has the same structure as SEQ ID NO: 1 except that the N-terminal 118 amino acid residues have been removed. The sequence shown in SEQ ID NO: 4 has a structure in which 147 amino acid residues on the N-terminal side of SEQ ID NO: 1 have been removed. The sequence shown in SEQ ID NO: 5 is the same as SEQ ID NO: 1.
This is a configuration in which 198 amino acid residues on the N-terminal side of are removed. The sequence shown in SEQ ID NO: 6 is the same as N in SEQ ID NO: 1.
In this configuration, the terminal 27 amino acid residues have been removed.

Further, the fusion protein according to the present invention is:
Proteins for expressing activity (in the examples,
A gene encoding SAK protein) and a gene encoding an amino acid sequence used as an anchor sequence are linked by a conventional method.

By incorporating this fusion protein into an arbitrarily selected vector (for example, pSG-TERM) and transforming it into an appropriate host as a plasmid, it is possible to obtain a microorganism having the target protein immobilized on the cell surface. it can. In addition, gene binding, amplification, transformation into a host, and the like can be performed by combining well-known methods in the field of genetic recombination research.

[0021]

EXAMPLE Using lactic acid bacteria for foods, staphylokinase (SAK), which is a secreted protein, and Lactococcus lactis NCDO763 were synthesized with the aim of creating an expression system on the cell surface via an anchor sequence. A fusion protein with the anchor sequence of the produced protease (763 protease) was constructed, and its function as an anchor was examined.

1. Material Escherichia coli (E. Coli) JM109 strain (Toyobo Co., Ltd.)
And Lactococcus lactis Y
IT2081 strain and Lactobacillus c.
asei ) YIT9029 strain was used as a host. E. coli is L-bro
At th, the lactic acid bacteria were cultured in M17-glucose medium. Plasmid pSG-TERM was used as a vector. PCR
PBE31SAK1 and pMQ9
080 was used.

2. Shuttle Vector pSG-TERM FIG. 1 is an explanatory diagram schematically showing the structure of the plasmid pSG-TERM. This plasmid is a shuttle vector pBE31 (Japanese Unexamined Patent Publication No.
No. 53861) of about 0.21 kb of Acc-S.
The maI fragment was deleted, and further, a promoter (Em promoter) derived from the erythromycin resistance gene and an S promoter derived from the 763 protease gene were added to the BamHI and SphI sites.
D sequence and signal sequence (763 protease signal se)
q.), multiple restriction enzyme recognition sequence (MCS), terminator sequence derived from 763 protease gene (763 protease ter
A DNA fragment of about 0.46 kb including the H.minator) was inserted to obtain pSG-TERM.

3. A 3.1 kb BglII fragment derived from pLP763 containing the C-terminal of 763 protease was cloned into the BamHI site of plasmid pUC119 of pMQ9080 E. coli,
9080.

4. Sequence and Synthesis of Each Primer A DNA primer having the following sequence was synthesized with a DNA synthesizer (Mo
del 392, Abride Biosystems). (1) SGSAK5 ATTAA GTTCG AAGGA GGAAG CGCCA TGCTC (2) SAKSAL1R AACTA TTTTG TCGAC TTTCT T (3) MK245 CTCCG CGTCG ACAAG AAGAC TTCGC TGCTT AAC (4) MK160 CTCCG CGTCG ACGCT GCAAC TGGCA AAACT TGG (5) MK127 TTCCG CGTCG ACTTG CAAAG TCTGA AAACG AAG (6) MK098 TTCCG CGTCG ACAAA GGCGG CGGTC AAGGT ACC (7) MK057 TTCCG CGTCG ACACG AGCAC GGATG ATACG ACT (8) MK038 TTCCG CGTCG ACTTA CCCAA GACAG GAGATGCATCACTAACT

[5] Production of DNA Fragment Gene DNA was amplified by a PCR method using KOD DNA polymerase (Toyobo Co., Ltd.). S
The amplification of the AK gene was performed using pBE31SAK1 as a template,
Between the primers SGSAK5 and SAKSAL1R,
This was performed by repeating one cycle of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 74 ° C. for 60 seconds, and repeating this 30 times.

The amplification of the anchor sequence was performed by using the DraI fragment of pMQ9080 as a template and primers MK245 and MK245.
K160, MK127, MK098, MK057, MK
038 and ANCSPH1 at 98 ° C for 15 seconds, 60
The cycle was performed at 30 ° C. for 5 seconds and at 74 ° C. for 30 seconds, and this was repeated 30 times.

6. Production of Recombinant Plasmid (1) Preparation of pSAK-NS The DNA fragment containing the SAK gene amplified by PCR was
After cleavage with NspV and SalI and purification, pSG-T
Inserted into the NspV-SalI site of ERM, pSA
K-NS was created. FIG. 2 is an explanatory view schematically showing the structure of the SAK-anchor sequence fusion protein gene.
As shown in FIG. 2A, pSAK-NS is located downstream of the promoter of the erythromycin resistance gene, and is composed of the SAK gene-derived SD sequence (SD) and the SAK signal sequence (signal s).
eq.). Since a SalI site was inserted on the 3 ′ side, C
It has a structure in which a SAK mature protein having two amino acid residues added to the end, a termination codon, and a terminator sequence derived from a lactic acid bacterium protease.

(2) pSAK245, pSAK160, p
SAK127, pSAK098, pSAK057, pS
Preparation of AK038 A DNA fragment containing an anchor sequence grown by PCR was
After digestion and purification with SalI and SphI, pSAK-N
By inserting into the SalI-SphI site of S,
pSAK245, pSAK160, pSAK127, p
SAK098, pSAK057 and pSAK038 were prepared. As shown in FIGS. 2b to 2g, these plasmids have a protease-derived anchor sequence from the C-terminus at 243, 158 from the C-terminus via two amino acid residues derived from the SalI site at the C-terminus of the SAK protein. , 12
It encodes a fusion protein in which 5,96,55,36 amino acid residues are linked. Regarding the obtained plasmids, pSAK245 was deposited as Biotechnological Laboratory No. 15909 and pSAK038 was deposited as Biotechnological Laboratories No. 15910, respectively, at the Biotechnology Research Institute on October 17, 2008. ing. For other plasmids,
Deposition of the desired base sequence of pSAK245 was judged to be obtained by excision, and thus no bacterial deposit was performed.

[7] Transformation into host First, the structure of the prepared plasmid DNA was confirmed using Escherichia coli as a host. Then, each plasmid DNA (pSAK245, pSAK1
60, pSAK127, pSAK098, pSAK05
7, pSAK038) was introduced into Lactococcus lactis YIT2018 strain by electroporation,
SAK-NS, SAK245, SAK160, SAK1
27, SAK098, SAK057, and SAK038 were obtained. The lactic acid bacteria clones prepared and analyzed in the present example are summarized in Table 1 above.

[0031]

[Table 1]

8. Confirmation of expression of SAK protein (1) Measurement of SAK activity The overnight culture broth was directly used as a culture solution fraction, and the cell pellet obtained by centrifuging the suspension was resuspended in M17-glucose medium to obtain a cell fraction. And 10 μl of each reaction solution (chro
mozyme PL 0.5mM, plasminogen 0.06units / ml, polysor
bate 80 0.01%, 0.1M Tris-HCl pH8.0) 100μl and 3
The reaction was carried out at 7 ° C., and the absorbance at 405 nm was measured with time using a microplate reader model 450 (manufactured by Bio Rad).

FIG. 3 is a diagram showing the results of measurement of SAK activity. FIG. 3A shows the culture solution fraction, and FIG. 3B shows the bacterial cell fraction.
The vertical axis indicates the absorbance, and the horizontal axis indicates the reaction time. As shown in FIG. 3, the activity was detected in all clones except that the activity was not observed only in the vector into which the SAK gene was not introduced.

(2) SDS-PAGE and Western blotting 1 m overnight culture of Lactococcus lactis clones
was centrifuged to obtain a culture supernatant and a cell fraction. The culture supernatant is 2 × SDS sample buffer 20 μl after TCA precipitation.
And heated. Similarly, the cell fraction was 2 × SDS sampl.
The resultant was suspended in 20 μl of e buffer and heated. These samples were subjected to SD on a Phast System 20% Phast gel (Pharmacia).
After S electrophoresis, the DNA was transferred to a PVDF membrane, and Western blotting was performed using an anti-SAK antiserum as a primary antibody and an alkaline phosphatase-labeled anti-rabbit IgG antibody as a secondary antibody.

FIG. 4 is an explanatory view schematically showing the results of Western blotting, in which the culture supernatant (s
up) and the cell fraction (cell) are shown in order. As shown in the figure, no SAK protein was detected in the vector (Vector) into which the SAK gene was not introduced. It is considered that the surface protein of the cells was extracted by the SDS sample buffer in the cell fraction. SA in the culture supernatant
Many bands having a small molecular weight, which are considered to have caused degradation of the K (fusion) protein, were detected.

(3) Detection of SAK (fusion) protein on bacterial cell surface using alkaline phosphatase-labeled antibody Each plasmid (pSAK245, pSAK160, pSAK160)
AK127, pSAK098, pSAK057, pSA
K038) was similarly introduced by electroporation, and the host bacterium was transformed into Lactobacillus casei.
us casei ).

1 ml of overnight culture of Lactococcus lactis and Lactobacillus casei clones
Was centrifuged to obtain a bacterial cell fraction. 50 mM of the obtained cells
Tris-HCl pH 7.4 After washing twice with 1 ml, 1% BSA / T
The suspension was suspended in 0.5 ml of BST, kept at room temperature for 30 to 60 minutes, suspended in 0.5 ml of TBST containing 1 μl of anti-SAK antiserum, kept at room temperature for 30 to 60 minutes, and washed three times with 0.5 ml of TBST. Thereafter, suspension was held for 30 to 60 minutes at room temperature and suspended in TBST 0.5 ml containing alkaline phosphatase labeled antibody Rabbit IgG antibody 0.25 [mu] l, washed 3 times with TBST 0.5 ml, in TBST as OD ₆₆₀ is 7.5 It became cloudy and was used as a sample.

A substrate solution (1 mg) was added to 10 μl of the obtained sample.
p-Nitrophenyl Phosphate / 10ml diethanolamine buffe
r) Add 100 μl, react at room temperature, and measure absorbance at 405 nm over time with microplate reader model 4
50 (manufactured by Bio Rad).

FIG. 5 is a diagram showing the results of detection of SAK (fusion) protein on the surface of bacterial cells using an alkaline phosphatase-labeled antibody, and FIG. 5A shows Lactococcus lactis.
( Lactococcus lactis ), Figure b shows Lactobacillus casei
( Lactobacillus casei ). The vertical axis indicates the absorbance, and the horizontal axis indicates the reaction time (min).

In the figure, the activity of the absorbance indicates the activity of alkaline phosphatase (AP) bound to the cells. That is, the rabbit antibody SA was added to the SAK expressed on the cell surface.
Since the K antibody binds and further binds to the rabbit IgG antibody labeled with alkaline phosphatase,
If the activity of alkaline phosphatase is high, the number of SAKs expressed on the surface of bacterial cells will be large. Therefore, the steeper the slope of the graph shown in the figure, the higher the AP activity and the higher the amount of AP-labeled anti-rabbit IgG antibody bound to the cells. This means that the clone has a large amount of rabbit anti-SAK antibody on the cell surface, that is, a large number of SAK protein epitopes displayed on the cell surface.

As shown in the figure, in any of Lactococcus lactis and Lactobacillus casei, those having a large number of amino acid residues have high AP activity, that is, high expression of SAK on the cell surface. confirmed. The 36 amino acid residues are L. Control with lactis (vector, N
S), the difference from L.S. Casei could not confirm the difference. This may be due to the possibility that the fusion protein is buried in the cell wall of the bacterial cell. Therefore, an anchor sequence preferably exceeding 36 amino acid residues is required.

[0042]

As described above, the present invention immobilizes various useful proteins on the surface of bacterial cells by utilizing an anchor sequence of a protease derived from Lactococcus lactis or a partial sequence of the anchor sequence. In addition, there is an effect that the useful protein is expressed. Therefore,
According to the present invention, various proteins can be immobilized using, for example, highly safe lactic acid bacteria, and thus it can be used for highly safe oral vaccines and the like.

[0043]

[Sequence list]

SEQ ID NO: 1 Sequence length: 243 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Organism name: Lactococcus lacti
s ) Strain name: NCDO763 strain Sequence Lys Lys Thr Ser Leu Leu Asn Gln Leu Gln Ser Val Lys Ala Ala Leu 1 5 10 15 Glu Thr Asp Leu Gly Asn Gln Thr Asp Ser Ser Thr Gly Lys Thr Phe 20 25 30 Thr Ala Ala Leu Asp Asp Leu Val Ala Gln Ala Gln Ala Gly Thr Gln 35 40 45 Thr Asp Asp Gln Leu Gln Ala Thr Leu Ala Lys Val Leu Asp Ala Val 50 55 60 Leu Ala Lys Leu Ala Glu Gly Ile Lys Ala Ala Thr Pro Ala Glu Val 65 70 75 80 Gly Asn Ala Lys Asp Ala Ala Thr Gly Lys Thr Trp Tyr Ala Asp Ile 85 90 95 Ala Asp Thr Leu Thr Ser Gly Gln Ala Ser Ala Asp Ala Ser Asp Lys 100 105 110 Leu Ala His Leu Gln Ala Leu Gln Ser Leu Lys Thr Lys Val Ala Ala 115 120 125 Ala Val Glu Ala Ala Lys Thr Val Gly Lys Gly Asp Gly Thr Thr Gly 130 135 140 Thr Ser Asp Lys Gly Gly Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly 145 150 155 160 Asp Ile Gly Lys Asp Lys Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly 165 170 175 Gly Asn Ile Pro Thr Asn Pro Ala Thr Thr Thr Ser Thr Ser Thr Asp 180 185 190 Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu 19 5 200 205 Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly 210 215 220 Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln 225 230 235 240 Arg Glu Glu 243

SEQ ID NO: 2 Sequence length: 158 Sequence type: amino acid Topology: Linear Sequence type: Peptide Origin Organism name: Lactococcus lacti
s ) Strain name: NCDO763 strain Sequence Ala Ala Thr Gly Lys Thr Trp Tyr Ala Asp Ile Ala Asp Thr Leu Thr 1 5 10 15 Ser Gly Gln Ala Ser Ala Asp Ala Ser Asp Lys Leu Ala His Leu Gln 20 25 30 Ala Leu Gln Ser Leu Lys Thr Lys Val Ala Ala Ala Val Glu Ala Ala 35 40 45 Lys Thr Val Gly Lys Gly Asp Gly Thr Thr Gly Thr Ser Asp Lys Gly 50 55 60 Gly Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly Asp Ile Gly Lys Asp 65 70 75 80 Lys Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly Gly Asn Ile Pro Thr 85 90 95 Asn Pro Ala Thr Thr Thr Ser Thr Ser Thr Asp Asp Thr Thr Asp Arg 100 105 110 Asn Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu Pro Lys Thr Gly Glu 115 120 125 Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly Val Ile Val Val Ser 130 135 140 Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln Arg Glu Glu 145 150 155 158

SEQ ID NO: 3 Sequence length: 125 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: Lactococcus lacti
s ) Strain name: NCDO763 strain Sequence Leu Gln Ser Leu Lys Thr Lys Val Ala Ala Ala Val Glu Ala Ala Lys 1 5 10 15 Thr Val Gly Lys Gly Asp Gly Thr Thr Gly Thr Ser Asp Lys Gly Gly 20 25 30 Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly Asp Ile Gly Lys Asp Lys 35 40 45 Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly Gly Asn Ile Pro Thr Asn 50 55 60 Pro Ala Thr Thr Thr Ser Thr Ser Thr Asp Asp Thr Thr Asp Arg Asn 65 70 75 80 Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu Pro Lys Thr Gly Glu Thr 85 90 95 Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly Val Ile Val Val Ser Leu 100 105 110 Met Gly Val Leu Gly Leu Lys Arg Lys Gln Arg Glu Glu 115 120 125

SEQ ID NO: 4 Sequence length: 96 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: Lactococcus lacti
s ) Strain name: NCDO763 strain Sequence Lys Gly Gly Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly Asp Ile Gly 1 5 10 15 Lys Asp Lys Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly Gly Asn Ile 20 25 30 Pro Thr Asn Pro Ala Thr Thr Thr Ser Thr Ser Thr Asp Asp Thr Thr 35 40 45 Asp Arg Asn Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu Pro Lys Thr 50 55 60 Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly Val Ile Val 65 70 75 80 Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln Arg Glu Glu 85 90 95 96

SEQ ID NO: 5 Sequence length: 55 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: Lactococcus lacti
s ) Strain name: NCDO763 strain Sequence Thr Ser Thr Asp Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly 1 5 10 15 Lys Gly Ala Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe 20 25 30 Gly Phe Leu Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu 35 40 45 Lys Arg Lys Gln Arg Glu Glu 50 55

SEQ ID NO: 6 Sequence length: 36 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: Lactococcus lacti
s ) Strain name: NCDO763 strain Sequence Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu 1 5 10 15 Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys 20 25 30 Gln Arg Glu Glu 35 36

[Brief description of the drawings]

FIG. 1 is an explanatory diagram schematically showing the structure of a plasmid pSG-TERM.

FIG. 2 is an explanatory view schematically showing the structure of a SAK-anchor sequence fusion protein gene.

FIG. 3 is a diagram showing the results of measuring SAK activity,
Fig. a shows the culture solution fraction, and Fig. b shows the bacterial cell fraction. The vertical axis indicates the absorbance, and the horizontal axis indicates the reaction time.

FIG. 4 is an explanatory diagram schematically showing the results of Western blotting, in which a culture supernatant (sup) and a cell fraction (cell) for each clone are shown in order.

FIG. 5 is a diagram showing the results of detection of SAK (fusion) protein on the surface of bacterial cells using an alkaline phosphatase-labeled antibody. FIG. 5A is a diagram showing the results of Lactococcus lactis.
us lactis ), and the figure b shows Lactobaci
llus casei ). The vertical axis indicates the absorbance, and the horizontal axis indicates the reaction time (min).

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12Q 1/68 C12Q 1/68 A (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 C12R 1:01) (C12P 21 / 72 C12R 1:01) (72) Inventor Mariko Kadota 1-1-1, Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Tomoyuki Sako 1-1-1, Higashi-Shimbashi, Minato-ku, Tokyo No. 19 Yakult Honsha

Claims (3)

[Claims] 1. Lactococcus lactis ( Lactococcus)
lactis ) a step of obtaining a fusion gene sequence linking the gene sequence encoding the anchor sequence of the 763 protease derived from the NCDO763 strain and the gene sequence encoding the protein of interest, and introducing the fusion gene sequence into a host bacterium. A method for immobilizing a protein on a cell surface, comprising: culturing a host cell into which the fusion gene sequence has been introduced, and immobilizing the target protein on the cell surface. . 2. The method according to claim 1, wherein the anchor sequence has at least the following amino acid sequence. Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln Arg Glu Glu 3. The protein according to claim 2, wherein the anchor sequence comprises an amino acid sequence obtained by partially removing the following amino acid sequence while retaining the amino acid sequence shown in claim 2. Immobilization method. Lys Lys Thr Ser Leu Leu Asn Gln Leu Gln Ser Val Lys Ala Ala Leu Glu Thr Asp Leu Gly Asn Gln Thr Asp Ser Ser Thr Gly Lys Thr Phe Thr Ala Ala Leu Asp Asp Leu Val Ala Gln Ala Gln Ala Gly Thr Gln Thr Asp Asp Gln Leu Gln Ala Thr Leu Ala Lys Val Leu Asp Ala Val Leu Ala Lys Leu Ala Glu Gly Ile Lys Ala Ala Thr Pro Ala Glu Val Gly Asn Ala Lys Asp Ala Ala Thr Gly Lys Thr Trp Tyr Ala Asp Ile Ala Asp Thr Leu Thr Ser Gly Gln Ala Ser Ala Asp Ala Ser Asp Lys Leu Ala His Leu Gln Ala Leu Gln Ser Leu Lys Thr Lys Val Ala Ala Ala Val Glu Ala Ala Lys Thr Val Gly Lys Gly Asp Gly Thr Thr Gly Thr Ser Asp Lys Gly Gly Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly Asp Ile Gly Lys Asp Lys Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly Gly Asn Ile Pro Thr Asn Pro Ala Thr Thr Thr Ser Ser Serr Thr Asp Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln Arg Glu Glu