JPH11172A - DNA, vector and microorganism containing peptide transport protein gene - Google Patents

Abstract

(57) [Summary] (Solution) Lactobacillus helveticus ( Lactoba
DNA containing a peptide transport protein gene derived from Cillus helveticus ). A vector having the ability to produce a peptide transport protein into which the DNA has been incorporated. A microorganism capable of producing a peptide transport protein, which is transformed with the vector, and a membrane vesicle containing the peptide transport protein prepared from a cell produced by culturing the microorganism and producing the peptide transport protein. The peptide transport protein produced by the microorganism transformed with the vector of the present invention has the property of specifically incorporating dipeptides and tripeptides, has high thermostability, and can be used even in a high temperature range. Can be.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DNA containing a peptide transport protein gene derived from lactic acid bacteria Lactobacillus helveticus . The present invention also relates to a vector having the ability to produce a peptide transport protein into which the DNA has been incorporated.
Furthermore, the present invention relates to a microorganism capable of producing a peptide transport protein transformed with this vector, and a membrane vesicle containing the peptide transport protein prepared from a cell produced by culturing the microorganism to produce the peptide transport protein.

[0002]

2. Description of the Related Art It is known that organisms produce various transport proteins due to the need to take in nutrients from outside the body. And various peptide transport proteins have been found so far (Mol.Microbiol., Vol. 16, p.
825, 1995). This peptide transport protein can be roughly classified into two types according to the acquisition form of the energy source consumed during transportation. The first type utilizes ATP (adenosine triphosphate) in a living body. This type of peptide transport protein is
It is called BC transport protein, and a famous review of Higgins is known (Annu. Rev. Cell Biol., Vol. 8, p. 6).
7, 1992). The second type belongs to the PTR family and is named by Steiner (Mo
l. Microbiol., vol. 16, p. 825, 1995). This peptide transport protein carries out peptide transport utilizing the difference in proton concentration inside and outside the cell (proton driving force). The second type, a peptide transport protein that utilizes proton motive force, has been found in many organisms, but they are derived from eukaryotes, and those derived from prokaryotes include lactoproteins. Cockas Lactis ( L
actococcus lactis ) (J. Biol. Chem., vol. 264, p. 1139).
1, 1994). However, Lactococcus lactis ( Lactoco
ccus lactis ) is a mesophilic lactic acid bacterium, and thus has a drawback that the peptide transport protein derived from Lactococcus lactis can be used only in the medium temperature range around 30 ° C.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have been intensively studying peptide transport proteins derived from prokaryotes, and found that peptide transport proteins can be produced in Lactobacillus helveticus. I found that there is. Therefore, Lactobacillus helveticus ( Lactobacillus h
elveticus ) to obtain a DNA capable of producing a peptide transport protein, and to transform Escherichia coli with a vector incorporating the DNA, a peptide having the property of specifically incorporating dipeptides and tripeptides. It was confirmed to produce a transport protein. Furthermore, they have found that dipeptides and tripeptides can be specifically incorporated by using a membrane vesicle containing a peptide transport protein prepared from Escherichia coli that has produced the peptide transport protein, and completed the present invention. Was. Accordingly, the present invention relates to Lactobacillus helveticu
s ) DNA containing peptide transport protein gene from
The task is to provide In addition, the present invention, Lactobacillus helveticus ( Lactobacillushelveticus )
An object of the present invention is to provide a vector capable of producing a peptide transport protein into which a DNA containing a gene derived from the peptide transport protein has been incorporated. Furthermore, the present invention provides a microorganism transformed with a vector capable of producing a peptide transport protein, and a membrane vesicle containing a peptide transport protein prepared from a cell produced by culturing the microorganism to produce the peptide transport protein. The task is to provide

[0004]

SUMMARY OF THE INVENTION Accordingly, the present invention provides
Lactobacillus helveticus (Lactobacillus helve
ticus ) -derived DN containing peptide transport protein gene
A. The present invention is also a DNA containing a peptide transport protein gene obtained by cutting a chromosomal gene of Lactobacillus helveticus with a restriction enzyme HpaI. The present invention also relates to Lactobacillus helveticus ( Lactobacillus h
elveticus ) and the DNA obtained by cutting the chromosomal gene with the restriction enzyme HpaI was subjected to inverse PCR (inverse PC
It is a DNA containing a peptide transport protein gene having an EcoRV site and a BamHI site obtained by amplification by the R) method. The present invention also relates to Lactobacillus helveticus ( Lactobacillus helveticus ),
Lactobacillus helveticus (Lactobacillus helve
ticus ) The above DNA which is SBT 2171 (FERM P-14381). The present invention is also a vector having the ability to produce a peptide transport protein into which the DNA has been incorporated. The present invention is also a microorganism transformed with the vector and capable of producing a peptide transport protein. The present invention is also a membrane vesicle containing a peptide transport protein prepared from a cell produced by culturing the microorganism to produce the peptide transport protein.

Hereinafter, the present invention will be described in detail.
Lactobacillus helveticus of the present invention ( Lactobacill
us helveticus ) -derived DNA containing a peptide transport protein gene can be obtained by the method of Delley et al. (Appl. Environ.
iol., vol. 56, p. 1967, 1990) and the like, by cutting the chromosomal gene of Lactobacillus helveticus with the restriction enzyme HpaI. In the present invention, Lactobacillus helveticus ( Lactobacillus helveticus ),
Specifically, Lactobacillus helveticus ( Lactoba
cillus helveticus ) SBT 2171 (FERM P-14381).
Further, the DNA thus obtained is amplified by an inverse PCR method to thereby obtain an EcoRV site and Ba.
DNA containing a peptide transport protein gene having an mHI site can be obtained. These DNAs are incorporated into a vector to obtain a vector having the ability to produce a peptide transport protein, and the resulting vector is used to transform a microorganism such as Escherichia coli or Bacillus subtilis. Microorganisms can be obtained.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide transport protein derived from Lactobacillus helveticus of the present invention has the property of specifically addressing dipeptides and tripeptides. Therefore, it contains a peptide-transport protein prepared from a microorganism transformed with a plasmid capable of producing a peptide-transport protein in which the DNA of the present invention is incorporated into a vector, or a cell produced by culturing these microorganisms to produce the peptide-transport protein. The resulting membrane vesicles can be used as peptide transport proteins. For example, the cells of these microorganisms themselves, or membrane vesicles prepared by removing the membrane components from the cells, can be used as a carrier for specifically enriching peptides, dipeptides and tripeptides, and as a carrier for dipeptides and tripeptides. It can be used for sensors etc. that detect Since Lactobacillus helveticus is a high-temperature lactic acid bacterium, the peptide transport protein of the present invention derived therefrom has a characteristic that it can be used even in a high temperature range of about 50 ° C. When using membrane vesicles as the peptide transport protein, a method using an ion gradient (Poolman et al., Handbook of Biomembrane, 1992)
And fusion with bovine myocardium-derived cytochrome C oxidase (Driessen et al., Proc. Natl. Acad. Sci., Vo
l.82, p.7555, 1985) and the like.

[0007]

Next, the present invention will be described in more detail with reference to Examples and Test Examples. Example 1 (a) Preparation of chromosomal gene of Lactobacillus helveticus The method of Delley et al. (Appl. Environ. Microbiol. Vol. 56, p.
p.1967-1970, 1970), a chromosomal gene of Lactobacillus helveticus was prepared. That is, Lactobacillus helveticus ( L
After culturing actobacillus helveticus ) SBT2171 (FERM P-14381) in MRS medium at 37 ° C for 16 hours, it was inoculated in 5% MRS medium and cultured at 37 ° C for 5 hours. The resulting culture is
After washing twice with 100 mM phosphate buffer (pH 7.0), the cells were collected by centrifugation, and suspended in 10 mM Tris buffer (pH 7.8) containing 1 mM EDTA (ethylenediaminetetraacetic acid).
Next, an operation for destroying the cells was performed. That is,
Protease K (manufactured by Boehringer Mannheim) was added to the cell suspension at a concentration of 250 μg / ml, and the concentration was 500 μg / ml.
Protease E (manufactured by Seikagaku Corporation) was added to each at a concentration of μg / ml, and after treatment at 37 ° C. for 30 minutes, 1 mM
The cells were washed with 10 mM Tris buffer (pH 7.8) containing EDTA, and the treated cells were suspended in the buffer. In this treated cell suspension,
Add 160 U of Mutanolysin (Seikagaku Corporation), 37 ℃, 30
Minutes, and then perform SDS so that the concentration becomes 0.1%.
(Sodium lauryl sulfate), EDTA to a concentration of 75 mM, and Protease K to a concentration of 200 μg / ml, respectively, and treatment at 65 ° C. for 2 hours. Then, the cell suspension subjected to the above treatment is washed three times with an organic solvent (phenol: chloroform = 1: 1),
After separating the aqueous layer with the same organic solvent to recover the aqueous layer, cold ethanol was added to the aqueous layer to a final concentration of 70%, and the chromosomal gene was recovered from the resulting precipitate by winding up the chromosomal gene with a sterile toothpick. (B) Preparation of DNA containing the peptide transporter protein gene The Lactobacillus helveticus (Lactobacillus h
Elveticus ) SBT 2171 (FERM P-14381) chromosomal gene was digested with the restriction enzyme HpaI at 37 ° C. for 6 hours to obtain DNA containing the peptide transport protein gene.

Example 2 (Amplification of DNA) DNA containing a peptide transport protein gene was amplified by inverse PCR. That is, the DNA obtained in Example 1 was ligated with T4-DNA ligase (Boehringer Mannheim) to form a circular DNA.
And used as a template for amplification. In addition, the amplification of DNA was carried out according to Table 1.
Was performed according to the composition shown in FIG.

[0009]

Table 1---------------------------------------------------------------------------------------------------------------------------------------------------------------- 10 μl 5 mM dNTP mix (Boehringer Mannheim) 4 μl 100 mM magnesium chloride 2 μl Primer 1 2 μl Primer 2 2 μl Sterile water 77 μl −−−−−−−−−−−−−−−−−−−−−−−−−− ----------- Primer 1: 5'-TCCTGTAATTGTTTTTATTG (EcoR V site is introduced) Primer 2: 5'-ATGACACATATTTCACTACTG (BamHI site is introduced)

Next, 1 μl of PWO polymerase (Boehringer Mannheim) was added, and the following reaction steps (1) to (5) were performed: (1) 95 ° C., 10 minutes, (2) 95 ° C., 90 seconds, (3) ) 50 ℃, 2
The DNA was amplified by repeating 30 cycles of (5) (2) to (4) for 30 minutes at (4) 72 ° C. for 3 minutes to obtain DNA containing a peptide transport protein gene. The nucleotide sequence of this DNA and the amino acid sequence deduced from the nucleotide sequence are shown in FIG.
3 and SEQ ID NO: 1 in the sequence listing. Immediately before the start of the reaction, sterilized mineral oil was overlaid to prevent evaporation of water.

Example 3 (Preparation of vector) DNA containing the amplified peptide transport protein gene was ligated to a vector. That is,
Escherichia coli vector pTAQI (manufactured by Gencor) previously cut with restriction enzymes BamHI and EcoRV, and the DNA obtained in Example 2 were mixed, and ligated with DNA ligase (manufactured by Boehringer Mannheim) to carry out peptide transport. A vector having a peptide transport protein-producing ability into which a DNA containing a protein gene was incorporated was obtained.

Example 4 (Transformation of Microorganism) Microorganisms were transformed using a vector capable of producing a peptide transport protein. That is, E. coli E1772 strain previously treated with calcium chloride
(J. Bacteriol., Vol. 173, pp. 234-244, 1991) and the vector having the ability to produce a peptide transport protein obtained in Example 3 were mixed, and the vector was subjected to heat shock at 42 ° C. for 90 seconds. It was introduced into Escherichia coli to obtain Escherichia coli capable of producing a peptide transport protein. The vector introduced into Escherichia coli was selected on a medium (peptone 10 g / l, yeast extract 5 g / l, salt 5 g / l) supplemented with 100 μg / ml ampicillin.

Test Example 1 (Measurement of Dipeptide Uptake by Escherichia coli) 10 mM D-lactic acid was added to Escherichia coli having the ability to produce a peptide transport protein obtained in Example 4, and oxygen was blown into the Escherichia coli. One minute later, ¹⁴ C-labeled prolylalanine (Pro-Al
a) was added, samples were collected over time, and E. coli was trapped with a membrane filter (0.45 μm). After washing the filter twice with ice-cooled 0.1 M lithium chloride, the filter was dissolved in a liquid scintillation cocktail, and the radioactivity was measured to determine the amount of dipeptide uptake by Escherichia coli. As controls, untransformed Escherichia coli and Lactococcus lactis prepared by the same method as in Example 4 were used.
A similar test was performed on Escherichia coli transformed with a vector capable of producing a peptide transport protein incorporating an NA fragment. FIG. 4 shows the results. According to the results shown in FIG. 4, untransformed Escherichia coli had a very low dipeptide uptake and hardly increased over time. In addition, Lactobacillus helveticus of the present invention ( Lactoba
cillus helveticus ) DNA is introduced into Lactococcus lactis DN.
It was found that the amount of dipeptide uptake was much higher than that of E. coli into which A was introduced.

Example 5 (Preparation of membrane vesicles) The Escherichia coli having the ability to produce a peptide transport protein obtained in Example 4 was cultured, and membrane vesicles were prepared from the cells that produced the peptide transport protein. That is, M9 medium supplemented with yeast extract 10 g / l and sodium succinate 10 g / l (disodium hydrogen phosphate 64 g / l, potassium dihydrogen phosphate 15 g / l, sodium chloride 2.5 g / l, ammonium chloride 5.0 g / l) M9 salt mix solution consisting of 200 l
After culturing at 37 ° C. for 3 hours with a 20% glucose solution (20 ml / l, 20% glucose solution), the culture was washed with potassium phosphate buffer (pH 7.0) and collected. And the method of Kaback (Methods of
According to Enzymology, vol. 22, pp. 99-120, 1971), membrane vesicles containing a peptide transport protein were prepared from this Escherichia coli. The prepared membrane vesicles were stored in liquid nitrogen until immediately before use in each test.

Test Example 2 (Measurement of Dipeptide Uptake by Membrane Vesicles) Phenazine methosulfate was added to the suspension of membrane vesicles containing the peptide transport protein obtained in Example 5 so that the final concentration was 50 μM. Was added, and air was blown in, and the temperature was maintained at 30 ° C. One minute later, potassium ascorbate was added to a final concentration of 10 mM, and one minute later, Pro-Ala labeled with ¹⁴ C was added. (0.45 μm) to trap E. coli. After the filter was washed twice with ice-cooled 0.1 M lithium chloride, the filter was dissolved in a liquid scintillation cocktail, and the radioactivity was measured to determine the amount of dipeptide incorporated into the membrane vesicles. Also, as a control,
A similar test was performed on membrane vesicles prepared from untransformed E. coli. FIG. 5 shows the results. From the results shown in FIG. 5, it can be seen that the membrane vesicles prepared from untransformed Escherichia coli have a low dipeptide uptake and little change over time, but the membrane vesicles of the present invention have a very high dipeptide uptake. It turned out to be high. Furthermore, the dipeptide uptake of the membrane vesicles of the present invention was compared at 37 ° C and 45 ° C. FIG. 6 shows the results. From FIG. 6, it was found that the membrane vesicles of the present invention had a higher activity in the high temperature range of 45 ° C. than in the case of 37 ° C.

[0016]

EFFECTS OF THE INVENTION Peptide transport proteins produced by microorganisms transformed with a vector into which a DNA containing the peptide transport protein gene of the present invention has been incorporated have the property of specifically incorporating dipeptides and tripeptides. Can be used to concentrate dipeptides and tripeptides, which are said to have good absorbency and high utilization efficiency. They can also be used as sensors for detecting dipeptides and tripeptides. In particular, the peptide transport protein produced by the present invention has high thermostability and activity even at a high temperature range of about 50 ° C., so it can be used online in a proteolytic system that requires an enzymatic reaction at a high temperature. It is useful as a concentrator and a sensor unit connected by.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of DNA containing the peptide transport protein-producing gene obtained in Example 2 and the amino acid sequence deduced from the nucleotide sequence.

FIG. 2 shows a continuation of FIG. 1 of the nucleotide sequence of the DNA containing the peptide transport protein producing gene obtained in Example 2 and the amino acid sequence deduced from the nucleotide sequence.

FIG. 3 shows a continuation of FIG. 2 of the nucleotide sequence of the DNA containing the peptide transport protein-producing gene obtained in Example 2 and the amino acid sequence deduced from the nucleotide sequence.

FIG. 4 is a graph showing the amount of dipeptide uptake by Escherichia coli in Test Example 1.

FIG. 5 is a graph showing the amount of dipeptide taken up by membrane vesicles in Test Example 2.

FIG. 6 is a graph showing the amount of dipeptide uptake of membrane vesicles at 37 ° C. and 45 ° C. in Test Example 2.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1884 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Lactobacillus helveticus Strain name: SBT 2171 Sequence AAGATGGACG TTAAGGTCAT CGAAAGCAAG GTTAACTTGA TCGAAGCTGA AAAAGATGCT -121 GTTAATGATG CAGTTGCTAA AGCAATTGAT TAAGTAATAT AAAGTAATAA AAATAAGGAT -61 CTATCTGTAA ATAGGATAGG TCCTTATTTT TCGTGGTGTA ATTGTTTTTA TTGCTTACCT -1 TAGATAAGAA AGGAGTTTTT CTTTGGGTAA ACGAGTTCAA ATACAGCATT CTTTGGACAG 60 CCAAAGGGCT TGTCCACTTT ATTCTTCACT GAAATGTGGG AGCGTTTCAG TTACTACGGC 120 ATG CGG GCT ATC TTA TTA TTC TAC 144 Met Arg Ala Ile Leu Leu Phe Tyr 1 5 ATG TAT TAC GCA GTT ACC AAA GGT GGT TTG GGG ATG TCT CAA ACT ACT 192 Met Tyr Tyr Ala Val Thr Lys Gly Gly Leu Gly Met Ser Gln Thr Thr 10 15 20 GCT GCT TCA ATC ATG TCG ATC TAT GGT TCG CTT GTT TAT TTA TCA ACA 240 Ala Ala Ser Ile Met Ser Ile Tyr Gly Ser Leu Val Tyr Leu Ser Thr 25 30 35 40 CTA GTT GGA GGC TGG CTT TCT GAC AGA GTA TGG GGC TCT AGA AAA ACA 288 Leu Val Gly Gly Trp Leu Ser Asp Arg Val Trp Gly Ser Arg Lys Thr 45 50 55 GTC TTC TAC GGC GGT GTG CTT ATT ATG TTG GGA CAC ATC GTT TTG GCT 336 Val Phe Tyr Gly Gly Val Leu Ile Met Leu Gly His Ile Val Leu Ala 60 65 70 TTG CCA GCT GGT GTA ACG GTG CTA TAC AGG TCG ATT GCT TTA ATC GTT 384 Leu Pro Ala Gly Val Thr Val Leu Tyr Arg Ser Ile Ala Leu Ile Val 75 80 85 GTA GGT ACT GGA TTA TTA AAG CCG AAC GTA TCC GAT ATG GTT GGG GGT 432 Val Gly Thr Gly Leu Leu Lys Pro Asn Val Ser Asp Met Val Gly Gly 90 95 100 CTT TAT TCG GTT GAA GAT CCC CGT CGT GAC GCT GGT TTC AGT ATT TTT 480 Leu Tyr Ser Val Glu Asp Pro Arg Arg Asp Ala Gly Phe Ser Ile Phe 105 110 115 120 GTT TTC GGG ATT AAC TTA GGC TCA ATT ATT GCT CCA TGG CTT GTA CCA 528 Val Phe Gly Ile Asn Leu Gly Ser Ile Ile Ala Pro Trp Leu Val Pro 125 130 135 TGG GCA GCT CAG GGC TTC GGT GTC CAT ATT TTT GGT AGC CAA TTG AAC 576 Trp Ala Ala Gln Gly Phe Gly Val His Ile Phe Gly Ser Gln Leu Asn 140 145 150 TTC CAT GCA GGA TTC TCA TTA GCT GCA GTT GGT ATG TTC TTT GGT TTA 624 Phe His Ala Gl y Phe Ser Leu Ala Ala Val Gly Met Phe Phe Gly Leu 155 160 165 GTA CAA TAT GTA CTT GGT GGT AAA AAA TAC TTA TCA ACT GAA AGT CTG 672 Val Gln Tyr Val Leu Gly Gly Lys Lys Tyr Leu Ser Thr Glu Ser Leu 170 175 180 ACA CCT AAT GAT CCT ATT GAT AAA GGC GAT TTG CTT AAT GTG ATC AAG 720 Thr Pro Asn Asp Pro Ile Asp Lys Gly Asp Leu Leu Asn Val Ile Lys 185 190 195 200 TGG GTT GTC ATT ATT ATC ATC GCA ATT GTT GCA ATT TTA GCC GCT ATG 768 Trp Val Val Ile Ile Ile Ile Ala Ile Val Ala Ile Leu Ala Ala Met 205 210 215 GCA GGA GTA GGG CAA TTA AGC GTT GAT AAT GTA ATT ACC TTA TTA ACT 816 Ala Gly Val Gly Gln Leu Ser Val Asp Asn Val Ile Thr Leu Leu Thr 220 225 230 ATT TTG GCG ATT GCA TTG CCA ATC TAC TAC TTC GTG ATG ATG TTT CGC 864 Ile Leu Ala Ile Ala Leu Pro Ile Tyr Tyr Phe Val Met Met Phe Arg 235 240 245 AGC TCA AAG GTT ACT AAG ATT GAG TTA GGA ATT CAT TTA CTA CCT GTA 912 Ser Ser Lys Val Thr Lys Ile Glu Leu Gly Ile His Leu Lue Pro Val 250 255 260 AGC TTG AAG AAT CGG CTG TTT TTC AAA AAA GGA TAT AAG CGG CTT AAG 960 Se r Leu Lys Asn Arg Leu Phe Phe Lys Lys Gly Tyr Lys Arg Leu Lys 265 270 275 280 CAG ATA ATT CAG CTT GAA CTT GCC ATA AAA AGG CAA AGC TTT ATT ATT 1008 Gln Ile Ile Gln Leu Glu Leu Ala Ile Lys Arg Gln Phe Ile Ile 285 290 295 CTG ATA GCG CTC ATC ATC ATG GCC AGC ATT TTG ATT CCA AAC AAA GTG 1056 Leu Ile Ala Leu Ile Ile Met Ala Ser Ile Leu Ile Pro Asn Lys Val 300 305 310 ATC ATT GCC AAA CAT CTG CTC AAG CTG GTT TTG TTG GTG TTC TAT TGG 1104 Ile Ile Ala Lys His Leu Leu Lys Leu Val Leu Leu Val Phe Tyr Trp 315 320 325 ATA GGT CTT AAT TTG ATC CCT TTT AGC ACA TTT GTT CTC TCC TTT CTT 1152 Ile Gly Leu Asn Leu Ile Pro Phe Ser Thr Phe Val Leu Ser Phe Leu 330 335 340 TTT TTA GAT TAT ATC AAA CAT ATG TTT AAG AAA GAG GGG GAA CAA GCA 1200 Phe Leu Asn Tyr Ile Lys His Met Phe Lys Lys Glu Gly Glu Gln Ala 345 350 355 360 AAA AAA ACA AAA GAA AAA AGC CGG ATT CAT CAC GGG ATT GAA ATC CCG 1248 Lys Lys Thr Lys Glu Lys Ser Arg Ile His His Gly Ile Glu Ile Pro 365 370 375 TTG TTT CTC CGG CAA TTA ATC ATA AAT ATA TTC ACC CTA ATA ATT TTG 1296 Leu Phe Leu Arg Gln Leu Ile Ile Asn Ile Phe Thr Leu Ile Ile Leu 380 385 390 GAA GGC GAA ACG CTT TTT GAT GAA AAT GGG GTA GAG GTC AAT ATT GCT 1344 Glu Gly Glu Thr Leu Phe Asp Glu Asn Gly Val Glu Val Asn Ile Ala 395 400 405 GAA CAT CCA GTG CAA GGA TAT ACG GAA TTG AAT ATC AAC CTT TTG AAT 1392 Glu His Pro Val Gln Gly Tyr Thr Glu Leu Asn Ile Asn Leu Leu Asn 410 415 420 AAA GAT AGC ATT GAT TTG TGG GCT GAT TGG ATT CAA AGC GTT GCA AAA 1440 Lys Asp Ser Ile Asp Leu Trp Ala Asp Trp Ile Gln Ser Val Ala Lys 425 430 435 440 TAT TTG CTT AAT ATC ATG TAT ACG GCA GAT GTG ATC GTA ATA ATT ATT 1488 Tyr Leu Lue Asn Ile Met Tyr Thr Ala Asp Val Ile Val Ile Ile Ile 445 450 455 TTC TAC CTC GTG AAG ATG GCG GCA TTG TGG TGG GCA TGG TCG TAT ATA 1536 Phe Tyr Leu Val Lys Met Ala Ala Leu Trp Trp Ala Trp Ser Tyr Ile 460 465 470 CCG TTG AGT ACA GTA TTT GTA GGC TAT AAA TAC TCA GGT AAA GAT GAG 1584 Pro Leu Ser Thr Val Phe Val Gly Tyr Lys Tyr Ser Gly Lys Asp Glu 475 480 485 TCC TTG CAA GCT GCT TTA GAA G TT TTA TGATAATGGA TCAAAGATTG 1631 Ser Leu Gln Ala Ala Leu Glu Val Leu 490 495 AATTAAAAAA CCTTCTCGCA ATGAGAAGGT TTTTCGTTAGTAG AATTCAGTAT GAATAATGTC 1691 ATCTTCTGGA CGATGTGCTT GG 1703

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1: 225) (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (7)

[Claims] [Claim 1] Lactobacillus helveticus (Lacto
DNA containing a peptide transport protein gene derived from Bacillus bacillus helveticus ). 2. The method of claim 1] Lactobacillus helveticus (Lacto
bacillus helveticus )
DNA containing a peptide transport protein gene obtained by digestion with aI. 3. A Lactobacillus helveticus (Lacto
bacillus helveticus )
The DNA fragment obtained by digestion with aI is
A DNA containing a peptide transport protein gene having an EcoRV site and a BamHI site obtained by amplification by the CR (inverse PCR) method. 4. The Lactobacillus helveticus (Lacto
bacillus helveticus ), Lactobacillus helveticus SBT 2171 (FERM P-1
The DNA according to any one of claims 1 to 3, which is 4381). 5. The DNA according to any one of claims 1 to 4,
A vector having the ability to produce a peptide transport protein into which is incorporated. 6. A microorganism capable of producing a peptide transport protein transformed with the vector according to claim 5. 7. A membrane vesicle containing a peptide transport protein prepared from cells produced by culturing the microorganism according to claim 6 to produce the peptide transport protein.