JPH025880A - Dna coding post prolyl peptidase - Google Patents

Abstract

PURPOSE:To obtain a recombinant DNA replicative in E.coli by preparing a structural gene coding a post prolyl peptidase existing in chromosome DNA of Bacteroides gingivalis and integrating the structural gene into E.coli vector. CONSTITUTION:Chromosome DNA of Bacteroides gingivalis is fragmentized and the obtained DNA fragment is integrated into a vector for E.coli to obtain a recombinant DNA. E.coli is transformed with the recombinant DNA and a strain capable of producing post prolyl peptidase is selected from the transformed E.coli. The above recombinant DNA is recovered from the selected E.coli. A DNA fragment containing a structural gene coding post prolyl peptidase can be produced by the above process.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a recombinant DNA incorporating a gene encoding post-prolyl peptidase produced by Bacteroides gingivalis. The present invention also relates to a method for producing the DNA, an E. coli containing the same, a method for producing post-prolyl peptidase using this E. coli, and a DNA fragment encoding post-prolyl peptidase.

[Prior Art and Problems] In recent years, it has come to be believed that infections with various oral bacteria are involved in the onset of adult periodontal disease.

In particular, Bacteroides gingivalis, a Durham-negative anaerobic bacillus, is frequently detected in patient lesions (χ) and has attracted attention as the main causative bacterium of periodontal disease [:/A. Throp et al., Journal Dental
Research (J, 5 lots et at, J, D
ENT, Res, 63:412.1984), such findings have provided major clues to the development of effective treatments for periodontal diseases. However, in the current situation where there is no effective treatment, it is desired to develop an accurate and rapid diagnostic method for early treatment of periodontal diseases. Several methods have been tried to improve the diagnosis of periodontal lesions. For example, bacteria may be isolated and cultured from periodontal pocket samples to identify the causative bacteria. However, it is difficult to say that this method can be widely used by the general public due to the difficulties in terms of equipment and the time and labor required when culturing anaerobic bacteria. It is also possible to use detection of enzyme activity thought to be associated with the causative bacteria. For example, Bacteroides gingivalis secretes a protease that hydrolyzes the peptide bond on the carboxyl side of proline, that is, post-prolyl peptidase, and detection of this enzyme activity can be applied to diagnostic methods. It is. Diagnostic methods using enzyme reactions are excellent in terms of time, labor, simplicity, and cost, but are still not satisfactory in terms of detection sensitivity.

In order to compensate for the drawbacks of the above-mentioned diagnostic methods, development of periodontal disease diagnostic methods using monoclonal antibodies and DNA probes has recently been proposed.

Generally, when producing a monoclonal antibody, a large amount of purified antigen is required, but it is extremely difficult and time-consuming to culture anaerobic bacteria in large quantities to prepare the antigen. Therefore, there is a problem that the target antigen cannot be obtained with high efficiency. Furthermore, in developing a diagnostic method using a DNA probe, it is necessary to isolate a DNA fragment that hybridizes (crosses over) with the chromosomal DNA of the desired causative bacterium.

In view of these circumstances, the present invention provides a method for highly efficiently producing post-prolyl peptidase produced by Bacteroides gingivalis, a Durum-negative anaerobic bacterium. The present invention also provides recombinant DNA and Escherichia coli used in this production method, and the recombinant DNA.
A manufacturing method is provided. Furthermore, the present invention
It provides a DNA fragment that hybridizes (crosses over) with the chromosomal DNA of Bacteroides gingivalis.

[Means for Solving the Problems] One aspect of the present invention is a structure encoding a protease that hydrolyzes a peptide bond on the carboxyl side of proline, that is, a post-prolyl peptidase, which is present in the chromosomal DNA of Bacteroides gingivalis. The present invention provides a recombinant DNA that can be replicated in E. coli by incorporating a gene into a vector for E. coli.

The present invention also provides a method for fragmenting Bacteroides gingivalis chromosome 1) NΔ, inserting the obtained DNA fragment into an E. coli vector to produce a recombinant DNA, and transforming E. coli with the recombinant DNA. The present invention provides a method for producing recombinant DNA, which comprises selecting a strain that produces post-prolyl peptidase from transformed E. coli and recovering the recombinant DNA from the selected E. coli.

Furthermore, the present invention provides an approximately 3.5 Kb Sau3AI cleavage fragment in the chromosomal DNA of Bacteroides gingivalis, and an approximately 2.9 Kb fragment excised by EcoRV.
The present invention provides a DNA fragment containing a structural gene encoding post-prolyl peptidase.

The recombinant DNA of the present invention is a vector for E. coli that has a structural gene encoding a post-prolyl peptidase derived from Bacteroides gingivalis, a Durum-negative anaerobic bacillus, and is replicable in E. coli. The structural gene encoding post-prolyl peptidase to be incorporated may be derived from any bacterial strain that is taxonomically identified as Bacteroides gingivalis. Examples of preferred strains include Bacteroides gingivalis strain ATCC 33277 and Bacteroides gingivalis strain 381 (Forsyth, USA).
(originated from Dental Center).

As a vector for E. coli, one that can be replicated in E. coli and has an appropriate marker can be used. Various vectors for E. coli are known and commercially available. Any of these known vectors can be used in the present invention. Suitable examples include pB11328, pt-t C79, etc.

The recombinant DNA of the present invention can be produced by a method consisting of the following five steps.

First Step In the first step, chromosomal DNA as a raw material is extracted from cells and fragmented. The chromosomal DNA used as the raw material is extracted from cells of Bacteroides gingivalis strain, and contains a gene encoding post-prolyl peptidase.

The operation for extracting chromosomal DNA from cells is performed using the known phenol method [K.
.

Biophys, Acta) 72. pp619-6
29 (1963)].

Fragmentation of the extracted DNA can be carried out using restriction enzymes or endonucleases that do not have base sequence specificity. Furthermore, by mechanical treatment, chromosome D
It is also possible to fragment the NA.

Second Step In the second step, the chromosomal DNA fragment obtained in the first step is incorporated into a DNA vector for E. coli to produce recombinant DNA. In the first step, when a restriction enzyme that produces cohesive ends is used, the above-mentioned DNA introduction vector is cut with the same restriction enzyme, and the chromosomal DNA obtained in the first step is
After annealing with the fragments, recombinant DNA can be created by ligating them with a DNA ligase such as T/l DNA ligase. In this case, in order to increase the efficiency of recombination, the DNA introduction vector is cut with a restriction enzyme, and then the well-known Ullrich (tJ 1lrich)
It is desirable to subject this to alkaline phosphatase treatment by the method of ) et al.

In addition, in the first step, if a restriction enzyme that produces blunt ends or an endonuclease without base sequence specificity is used, or if chromosome 1) NΔ is fragmented by mechanical treatment, an appropriate restriction enzyme is added to the end. linker cut by
It can also be ligated with a vector DNA fragment.

Third Step In the third step, E. coli is transformed with the recombinant DNA obtained in the second step. Generally, transformation can be performed by known competent cell methods.

In addition, the chromosomal DNA fragment obtained in the first step is
In the case of a large DNA fragment of approximately 0 Kb, the recombinant DNA is incorporated into the head of a lambda phage using the lambda in vitro packaging method, and E. coli is infected with this phage particle. can be introduced into cells. In this case, the so-called Kusmid vector, which has a cos site derived from lambda phage DNA in the vector DNA, is used as the vector for E. coli. For example, pIIC79 can be mentioned.

Fourth step In the fourth step, among the transformed bacteria, D containing the post-prolyl peptidase gene in the raw chromosomal DNA
Bacteria that are transformed with the recombinant DNA containing the NA fragment and thereby produce significant amounts of post-prolyl peptidase from Bacteroides gingivalis are selected.

First, depending on the marker of the DNA introduction vector used,
A primary selection is made for cells containing recombinant DNA. When pBrt322 and pi (C79) are used as vectors, the bacteria are cultured in soil containing tetracycline or ampicillin to obtain tetracycline-resistant bacteria or ampicillin-resistant bacteria.

Next, the antibiotic-resistant bacteria thus obtained are inoculated onto a medium also containing the antibiotic, and this is used as a master plate. For the recombinant E. coli clones grown on Master Plate 2, a portion of each colony was picked one by one, each clone was suspended in a buffer solution containing lysodeme, and then subjected to a freeze-thaw method. and lyse it. In addition, as a method for lysing the bacteria, mechanical crushing methods such as ultrasonic treatment can be used.

By measuring the bosuprolyl peptidase activity in the Escherichia coli lysate thus obtained, we can identify the recombinant DNA containing the DNA fragment containing the post-prolyl peptidase gene in the target raw chromosomal DNA. Secondary selection can be done.

Post-prolyl peptidase activity is measured using a known synthetic substrate, and the substrate used has a chromogenic group on the carboxyl side of proline, such as β-naphthylamine or p-nitroaniline. Any of these is fine as long as it has an amide bond.

Examples include glycyl-proline-4-methoxy-β-naphthylamide hydrochloride and Nα-penzoyl-arginylglycyl-phenylalanyl-broline-4-methoxy-β-naphthylamide. The substrate concentration in the reaction solution is usually 0.05 to 2mM, preferably 0.2mM.
It is about mM.

Fifth step In the fifth step, recombinant DNA is collected from the bacteria secondarily selected in the fourth step. This recovery can be easily carried out by the phenol method and density gradient centrifugation method described above.

By further subcloning the recombinant DNA obtained in the fifth step of subcloning, unnecessary DNA parts can be cut off, the recombinant DNA can be stably retained within cells, and the amount of enzyme production can be further increased. . Subcloning is performed by cleaving the obtained recombinant DNA with an appropriate restriction enzyme, inserting it into an E. coli vector in the same manner as above, transforming it, selecting for free, and recovering the recombinant DNA. Be able to do it. Any restriction enzyme can be used to cleave the recombinant DNA, which is the starting material for subcloning, as long as it has a cleavage site in the structural gene portion of the enzyme. Subcloning can be performed several times, and each subcloning can be performed by cutting into smaller pieces.

By transforming E. coli with the recombinant DNA obtained in the fifth step of 119 or the subsequent subcloning, bacteria that produce post-prolyl peptidase derived from Bacteroides gingivalis can be obtained. In addition, Bacteroides gingivalis D present on the recombinant DNA obtained by subcloning
The NA fragment has restriction enzyme cleavage sites as shown in the representative restriction enzyme map in FIG. That is, the Bacteroides gingivalis DNA fragment present on the recombinant DNA obtained by subcloning is approximately 3.5 Kb.
The size of Sau3A from the I3amHI cleavage site
It includes up to the I cleavage site. In addition, the restriction enzyme cleavage site is approximately 30 bp away from the BamH1 site.
inc11 site, about 150 bp and about 3050 bp), an EcoRV site about 900 bp apart, a Pvu11 site about 900 bp apart, and an A about 1650 bp apart.
at1 site, about 1800 bp apart, Pvu1 site, about 2160 bp apart, Bgl site, about 2
Hind [1 site and Hind
A Kpn1 site exists near the dln site.

Post-prolyl peptidase can be produced by culturing this strain and recovering the enzyme from the bacterial cells. E. coli can be cultured by known methods. For example, suitable carbon sources 1. This can be carried out by culturing with shaking in a medium containing a nitrogen source and trace amounts of metal elements. As the carbon source, glucose, glycerin, etc. can be used. The concentration of carbon source is usually O1l
The amount is about 10 to 10% by weight, preferably about 2% by weight.

As a nitrogen source, in addition to organic nitrogen such as peptone, polypeptone, and meat extract, inorganic nitrogen such as ammonium sulfate and ammonium chloride can be used. The concentration of the nitrogen source is about 0.5 to 5% by weight, preferably about 2% by weight. Furthermore, in order to prevent the recombinant DNA from falling off, an antibiotic corresponding to the drug resistance marker of the plasmid vector used may be added to the medium. The vector used was pHC79.
In the case of
About μ9/IIQ or tetracycline, preferably 1
It is possible to add about 0μ9/112. In addition, when the vector used is pI311328, in addition to the above-mentioned antibiotics, chloramphenicol is preferably used.
It is possible to add about 0 μg/mQ.

By culturing Escherichia coli transformed with recombinant D'NA in a medium as described above, post-prolyl peptidase derived from Bacteroides gingivalis can be accumulated within the cells.

The obtained culture solution was centrifuged at low speed (5000r, p, m,
, 20 minutes), collect the bacterial cells, suspend the bacterial cells in a buffer solution, and then crush or lyse the bacterial cells to obtain the post-prolyl peptidase from the bacterial intracellular extract obtained. Known methods can be used for disrupting or lysing bacterial cells. Examples include ultrasonic disruption, bacteriolysis using lysozyme, and freeze-thaw methods.

[Example] Examples of the present invention will be described below. All restriction enzymes used in the examples are commercially available from Toyobo Co., Ltd.

Example 1 Preparation of psN1 recombinant DNA and Escherichia coli strain harboring it Bacteroides nonsyvaris strain 381 was mixed with anerobic BH [medium 1f2 (2% by weight of peptone, 1% by weight of yeast extract, 0.0005% by weight of hemin, 0.05% by weight of cysteine). 05
Weight %, trace amounts of vitamin K and inorganic salts, purified water residue, p
) (7,0) under anaerobic conditions at 37°C, the bacteria were collected, and washed once with physiological saline (085 weight percent). Chromosomal DNA was extracted from the thus obtained bacterial cells by the phenol method, and further purified by cesium chloride equilibrium density gradient centrifugation to obtain 500μ9 chromosomal DNA.

After treating 500 μ9 of this DNA with Sau3AI for 15 minutes at 37°C, the treated solution was subjected to 10 to 40% sucrose density gradient centrifugation (24,00 Or, p, m, 40 hours). Both portions containing DNA fragments were recovered. 10μ7 cosmid vector ptlc79 was transformed into BamH
After treatment with I at 37°C for 3 hours, alkaline phosphatase treatment was performed according to a conventional method. This vector DNA fragment 10
μ9 and 10 μ9 of the above-mentioned approximately 40 Kb chromosomal DNA fragment were mixed and treated with T4 ligase at 10° CI for 6 hours to prepare recombinant DNA.

Using 1 μ9 of the obtained recombinant DNA, lambda in vitro packaging, that is, introduction of the recombinant DNA into the head of a lambda phage particle, was carried out according to a conventional method. For lambdyne in vitro packaging, a commercially available packaging kit [Amersham International, Inc.
ional plc, )] according to the method described in the kit.

The thus obtained lambda phage particles incorporating the recombinant DNA into the head were transformed into Escherichia coli 11BIOI using a conventional method.
The recombinant DNA was introduced into E. coli cells by infecting E. coli with B.

For primary selection of recombinant DNA-carrying bacteria, 50μq/
Agar medium containing mQn degree of ampicillin (agar 15% by weight)
, peptone 1% by weight, yeast extract 0°5% by weight, salt 1%
The cells were cultured at 30°C for 136 hours in 1% water (weight 1%, residual purified water).

Each colony of the grown recombinant DNA-carrying bacteria was inoculated into a medium having the same composition as above to prepare a master plate. A portion of each colony of recombinant DNA-carrying bacteria on the master plate was picked, and 100μy/IIu1
The cells were suspended in 1M Tris-HCl buffer (0.1-Q, pi-17.0) containing a high degree of zodim, and the cells were lysed by freezing and thawing according to a conventional method.

80 μQ of each of the obtained bacteriolytic solutions and 1 mM glycylproline-4-methoxy-β-naphthylamide hydrochloride solution 2
0μQ was mixed in a 96-well microtiter plate and reacted at 37°C for 4 hours, followed by stopping with 70μQ, coloring solution (Fast Garnene 1-G BC reagent 0.5 wt. 61%, Tween 20 200 wt. 0.1M acetic acid-sodium hydroxide pI (7°0) was added to select a strain showing significant enzyme activity (the strain in which the reaction solution turned red), and one strain was obtained.This strain was transformed into E. coli 1-IB. 101/pSN
1, and was deposited with the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology (Feikokuken) on May 28, 1986.
No. 0043). The mycological properties of this bacterium are the same as those of the known Escherichia coli t+Btot strain, except that it has extremely high post-prolyl peptidase activity.

DNA was extracted from E. coli 1-II3101/pSNl by the phenol method, and recombinant DNA was extracted by density gradient centrifugation.
A large amount of the recombinant DNA was collected and this recombinant DNA was named pSNl. The size of PSNI is approximately 43Kb.

Example 2 Preparation of pSN2 recombinant DNA and E. coli strain harboring it 10) Treat t9 psNt with BamHI at 37°C for 3 hours, and subject this I3amHI treated product to agarose gel electrophoresis to separate DNA fragments. did. The largest DNA fragment of 17 Kb among the detected DNA fragments was recovered from the gel according to a conventional method and self-ligated using T4 ligase.

Using the obtained recombinant DNA, E. coli strain H8101 was transformed by the competent cell method. 50μ9/
For an E. coli strain containing recombinant DNA grown on a medium containing 1 m of ampicillin, Example 1
Post-prolyl peptidase activity was examined according to the method described in . Significant enzyme activity was observed in all colonies examined. E. coli carrying this 17 Kb recombinant DNA was named HB101/pSN2, and on May 2, 1988,
Deposited with the Institute of Fine Technology on the 8th (National Institute of Fine Technology Deposit No. 10044)
.

The mycological properties of this bacterium are similar to those of the known Escherichia coli 11r except that it has extremely high post-prolyl peptidase activity.
The mycological properties were the same as those of the 3101 strain.

I) NA was extracted from Escherichia coli 1-+1310 l/pSN2 by the phenol method, and the recombinant 1) NA was collected in Okawa by density gradient centrifugation. This recombinant DNA was named pSN2.

Example 3 Preparation of pSN3 recombinant DNA and E. coli strain retaining it After treating 50 μ9 of pSN2 with Sau3AI at 37°C for 80 minutes, the treated solution was subjected to 1O to 40% sugar density gradient centrifugation (
24,00 Or, p, m 140 hours), then 3~
In step 5, both portions containing DNA fragments of size b were recovered. or,
10 μ9 of pBf1328 was treated with BamHI at 37° C. for 3 hours. After that, it was treated with alkaline phosphatase according to a conventional method, mixed with the above Sau3AI-treated DNA fragment, and recombinant D was prepared using T4 ligase.
Got NA. Using the recombinant DNA thus obtained, Escherichia coli 1-HB101 was purified by the competent cell method.
The strain was transformed, and the transformant was grown on a medium containing 50 μg/shoulder Qa of ampicillin and 10μ9/II (1 concentration) of chloramphenicol. The post-prolyl peptidase activity was investigated according to the method of 2010, and a strain with significant enzyme activity was selected. This strain was named Escherichia coli HB101/pSN3 and was deposited at the Microtech Institute on May 28, 1988 ( Bacteria No. 10045).

The mycological properties of this bacterium are similar to known Escherichia coli [-In2] except that it has extremely high post-prolyl peptidase activity.
The mycological properties were the same as those of the O2 strain.

DNA was extracted from E. coli 1-HB101/PSN2 by the phenol method, and a large amount of recombinant DNA was collected by density gradient centrifugation. This recombinant DNA was named pSN3. Figure 1 shows a restriction enzyme map of recombinant NAPSN4. In the figure, the white part is the chromosome fragment of Batateroides gingivalis 381, and the black part is vector pBR328. The size of pSN3 is 8.4 Kb, and there are 3.5 Kb in the Bamt11 cleavage site of pB11328.
D derived from Batateroides gingivalis strain 381 of Kb
Contains an NA fragment.

Example 4 Preparation of psNtI recombinant DNA and E. coli strain harboring it 10μ9 of pSN3 was treated with EcoRV at 37°C for 3 hours, and then DNA fragments were separated by agarose gel electrophoresis. Of the two DNA fragments detected, 2.
A small 9 Kb fragment was recovered from the gel according to a conventional method. The DNA fragment thus obtained was ligated with EcoflV-treated and alkaline phosphatase-treated pBrt328 using T4 ligase to create a recombinant 1) NA
was created. Using this recombinant 1) NA, Escherichia coli 11101 was transformed by the competent cell method. Transformants were grown in the same medium as above. The grown transformants were examined for post-prolyl peptidase activity according to the method of Example 1, and bacteria with significant enzyme activity were selected. This strain was transformed into E. coli f (B
Named 101/psN I l, May 2, 1986
It was deposited with the Microtech Institute on the 8th (Fiber Science Institute Deposit No. 10046).

This bacteriological property of E. coli 1-r is similar to that of the known E. coli 1-r except for extremely high post-prolyl peptidase activity.
The mycological properties of the BloI strain are the same.

1) NA was extracted from Escherichia coli HBIOl/pSNl by the phenol method, and the recombinant was recovered by density gradient centrifugation.

FIG. 2 schematically shows this operation using a restriction enzyme map. In the figure, the white part is the chromosome fragment of Bacteroides gingivalis strain 381, and the black part is vector pBR328.

What is the size of psN11? , 8 Kb, pBR328
A 2.9 Kb DNA fragment derived from Bacteroides nonsyvaris strain 381 is incorporated into the Ecol'tV cleavage site.

Example 5 Production of post-prolyl peptidase Escherichia coli 1-IBIOI/pSN thus obtained
3 and 1. The post-prolyl peptidase productivity of lI'3101/pSN I1 was investigated.

Each strain was inoculated into IQ broth (peptone 1%, yeast extract 05% by weight, salt 1% by weight, purified water remaining),
The cells were cultured with shaking at 37°C and collected. 0 bacterial cells
．． After suspension in 01M Tris-HCl buffer (pl7,5), ultrasonication (100W).

2 minutes), centrifugation (12,000 r, +),
The microbial cell debris was removed (20 minutes), and the resulting clear liquid was examined for post-prolyl peptidase activity.

In addition, as a control, E. coli) [B101/pr3
r (328 strain was cultured and the same procedure as above was performed, and the resulting supernatant was examined for enzyme activity.

Purine-proline-4methoxy-β-naphthylamide hydrochloride was used as a substrate for the enzyme reaction.

The reaction conditions were as follows: 80μg of 1mM substrate was added to 320μQ of intracellular enzyme extract, and after reaction at 37°C for 2 hours, stop and color development solution (
Add 160 μQ of Fast Garnet GBC reagent 0.5% by weight, Tween 20 20%, 0.1M acetic acid-sodium hydroxide pi-17,0), and measure the amount of 4-methoxy β-naphthylamine liberated by the enzymatic reaction by spectroscopy. Measured with a photometer (0, D, 5ss).

The results are shown in Table 1. In addition, in the table, 1μ per hour at 37℃
The amount of enzyme that liberates 4-methoxy-β-naphthylamine from M was defined as 1 unit (U).

Table 1 [Effects of the Invention] According to the present invention, post-prolyl peptidase secreted extracellularly by Bacteroides noncivaris can be produced with high efficiency. In addition, a DNA fragment containing a structural gene encoding a post-prolyl peptidase of approximately 2.9 Kb, which is cleavable in the Sau3AI cleavage fragment of 1) at approximately 3°5 in the chromosomal DNA of Bacteroides nonsyvaris, is excised by EcoRV. Can you get it?

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme cleavage map of psN3, Figure 2 is a restriction enzyme cleavage map of psN3.
Restriction enzyme cleavage map of pSNII schematically showing the method for preparing sN11.

Claims (1)

[Scope of Claims] (1) A protease (hereinafter referred to as post-protease) that specifically hydrolyzes the peptide bond on the carboxyl side of proline in a proline-containing peptide that is present in the chromosomal DNA of Bacteroides gingivalis. A recombinant DNA that can be replicated in E. coli by incorporating a gene encoding Rilupeptidase into an E. coli vector.
A. (2) The recombinant DNA according to claim (1), which is pSN3. (3) The recombinant DNA according to claim (1), which is pSN11. (4) (a) Chromosome D of Bacteroides gingivalis
Fragment the NA, (b) Incorporate the obtained DNA fragment into an E. coli vector to produce a recombinant DNA, (c) Transform E. coli with the recombinant DNA, (d) Transform the E. coli. A method for producing recombinant DNA, which comprises selecting a strain of E. coli that produces post-prolyl peptidase, and (e) recovering the recombinant DNA from the selected E. coli. (5) Perform the transformation in steps (b) and (c) by ligating the obtained DNA fragment to a cosmid vector for E. coli,
The method according to claim (4), which is carried out by introducing recombinant DNA into lambda phage particles and infecting Escherichia coli with the thus obtained lambda phage. (6) The method according to claim (5), wherein the cosmid vector is pHC79. (7) The method according to any one of claims (4) to (6), further comprising subcloning the obtained recombinant DNA. (8) Perform subcloning three times, and for the first subcloning, use BamH I to clone the raw recombinant DNA.
The second subcloning was performed by cutting the DNA fragment of about 17 Kb and self-ligating the DNA fragment of about 17 Kb.
The third subcloning was performed by partially cleaving the DNA using EcoRV and collecting approximately 4 Kb DNA fragments and incorporating them into an E. coli vector.
．． The method according to claim (7), which is carried out by collecting 9 Kb DNA fragments and incorporating them into an E. coli vector. (9) Escherichia coli containing a recombinant DNA capable of replicating in Escherichia coli in which a gene encoding post-prolyl peptidase derived from Bacteroides gingivalis is incorporated into an Escherichia coli vector. (10) E. coli HB101/pS
N3 or HB101/pSN11 (Feikoken Bacteria 100th
Claim No. 45 or No. 10046)
E. coli as described in section 9). (11) Recombinant DNA that can be replicated in E. coli by incorporating a gene encoding post-prolyl peptidase derived from Bacteroides gingivalis into an E. coli vector
1. A method for producing post-prolyl peptidase, which comprises culturing E. coli containing said post-prolyl peptidase, and recovering said post-prolyl peptidase from the culture. (12) (a) Fragment the chromosomal DNA of Bacteroides gingivalis, (b) Incorporate the obtained DNA fragment into a vector for E. coli to produce recombinant DNA, and (c) Infect E. coli with the recombinant DNA. (d) selecting a strain that produces post-prolyl peptidase from the transformed E. coli, and (e) culturing the selected E. coli and producing post-prolyl peptidase derived from Bacteroides gingivalis from the culture. A method for producing post-prolyl peptidase, which comprises recovering post-prolyl peptidase. (13) The transformation in steps (b) and (c) was carried out by ligating the obtained DNA fragment to a cosmid vector for E. coli and introducing it into lambda phage particles to produce a recombinant DNA. The method according to claim (12), which is carried out by infecting E. coli with lambda phage. (14) The method according to claim (13), wherein the cosmid vector is pHC79. (15) After step (d), the obtained recombinant DNA is further subcloned.
The method described in section. (16) The method according to claim (15), wherein the E. coli containing the recombinant DNA is E. coli HB101/pSN3 or HB101/pSN11 (Feikoken Bibori No. 10045 or No. 10046). . (17) Chromosomal DNA of Bacteroides gingivalis
D, which contains the structural gene encoding the approximately 2.9 Kb post-prolyl peptidase that is present in the approximately 3.5 Kb Sau3A I cleavage fragment and excised by EcoV.
NA fragment.