JPH084510B2 - DNA encoding the BanI restriction endonuclease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a DN encoding a BanI restriction endonuclease.
A, regarding.

[Conventional technology]

So far, about 120 types of type II restriction enzymes have been discovered from bacteria and commercialized as type II restriction enzymes. The type II restriction enzyme is an enzyme with a very high specificity that recognizes a specific base sequence in a deoxyribonucleic acid (DNA) chain and cleaves it, and is widely used in the field of genetic engineering.
It is also expected to be used for clinical tests by genetic diagnosis. BanI restriction endonuclease is one of the type II restriction enzymes. This enzyme is an enzyme that recognizes and cleaves GGPyPuCC in the nucleotide sequence of DNA, and is an enzyme produced in Bacillus aneurinolyticus IAM1077. This Bacillus aneurinolyticus IAM1077
Other than BanI, other restriction enzymes BanII and BanIII were produced at the same time in the bacterial cells, and it was very difficult to remove them, and there was a manufacturing problem.

On the other hand, the restriction site methylase methylates the recognition base sequence of the restriction enzyme, and it is used in gene manipulation to prevent the restriction enzyme from cleaving by methylating the recognition sequence of the restriction enzyme. However, up to now, no BanI methylase that methylates the recognition base sequence of BanI restriction endonuclease and its DNA have been found.

[Problems to be Solved by the Invention]

BanI is applied to hosts other than Bacillus aneurinolyticus using genetic engineering technology
If you can produce a restriction endonuclease,
The Bacillus aneurin
The problem of contaminating enzymes derived from
It is possible to obtain BanI restriction endonuclease.
An object of the present invention is to provide a DNA coding for the enzyme which is first required when producing a BanI restriction endonuclease by this genetic engineering technique.

[Means for solving the problem]

The present inventors collected chromosomal DNA from BanI restriction endonuclease-producing bacterium, digested the chromosomal DNA obtained with an appropriate restriction enzyme, and integrated it into a plasmid such as pBR322. The BanI restriction endonuclease and the BanI methylase-producing bacterium are each isolated by growing the bacterium, and the BanI restriction endonuclease and the BanI methylase are collected by culturing these producing bacteria and the enzymes are encoded.
The inventors have succeeded in isolating and identifying DNA, and have completed the present invention.

That is, the present invention provides (1) Bacillus aneinolyticus (Bacillus aneuri)
DNA fragment encoding a BanI restriction endonuclease derived from Nolyticus) and having a base sequence encoding the amino acid sequence represented by Formula I below.

Formula I (2) An expression vector containing the DNA fragment according to (1).

About.

 The present invention is described in detail below.

The plasmid containing the BanI restriction endonuclease gene of the present invention has a form capable of growing in cultured cells such as colicin E1 factor known as an extrachromosomal gene (plasmid) of a microorganism belonging to the genus Esheri-chia. A plasmid comprising a BanI restriction endonuclease gene derived from Bacillus aneurinolyticus IAM1077, a DNA fragment containing the BanI methylase gene, and a DNA fragment containing only the BanI methylase in the plasmid, wherein the vector DNA
In addition to those extracted from naturally occurring, it may be partially deleted in a portion of the DNA other than the essential part for proliferation ColE1 strain, pBR322 strain, pSC101 strain, p
Examples include MB9 strains and lambda phage strains.

As a method for incorporating the chromosomal DNA fragment into the vector DNA, any known method can be applied. For example, a method is used in which chromosomal DNA is cleaved at a specific site by treatment with an appropriate restriction enzyme, then mixed with vector DNA treated in the same manner and religated with ligase.

The pBR322 plasmid was used as the vector DNA and the Bacillus anoillinolyticus
cus) IAM1077, a novel plasmid pBANIRM 8 can be obtained by incorporating the chromosomal DNA fragment. p
The restriction enzyme map of BANIRM 8 plasmid is shown in FIG.
As shown in FIG. 1, this plasmid was found in the restriction site of pBR322 plasmid, Bacillus anoillinolyticus.
DNA containing the BanI restriction endonuclease gene and BanI methylase gene of IAM1077
It is a plasmid having an 8 kb base pair in which a fragment is integrated.

The BanI restriction endonuclease gene of the invention, and
The BanI methylase gene can be identified as follows.

First, the present inventors used the present plasmid strain Escherichia coli HB101 (pBanIRM8) (FERM P- to identify the BanI restriction endonuclease gene.
9424) [Escherichia coli H
It is the same as B101 (pBAN11). ] Was cultured and the bacterial cells were obtained.

BanI restriction endonuclease and BanI methylase were purified and purified from the cells by electrophoresis (SDS PAGE) to obtain a single band, and the N-terminal amino acid sequence of this sample was determined by an amino acid sequencer.

Further, the above strain producing BanI restriction endonuclease was cultured, and a plasmid was prepared from the cultured bacterial cell. This plasmid is naturally a DNA encoding BanI restriction endonuclease and a DNA encoding BanI methylase.
, Which can be used to determine the base sequence.
In order to determine the DNA base sequence, a DNA fragment in the region that seems to encode the above two enzymes from this plasmid was transferred to a vector suitable for base sequence determination, for example, pUC18, M13mp18, and the M13-dioxy method (Sanger, Pro. Natl.Acad.Sci.74,5
Determined from 463-5467 (1977). That is, pUC18, M13mp18 after changing to a recombinant plasmid, by determining the restriction enzyme site by various restriction enzymes, and by the cloning method using subcloning, magbean nuclease, exonuclease III, to create various deletion mutants BanI restriction endonuclease The region of the gene, the BanI methylase gene, was determined. Furthermore, the nucleotide sequence of the insertion mutant DNA fragment was determined. In addition, in the deletion mutant in which only the former one of the restriction enzyme activity and the methylase activity is expressed, the bacterium is killed, and the base sequence may not be determined because the DNA in the region where the base sequence should be determined cannot be obtained. In such a case, it was determined by utilizing the custom primer method or the like.

As a result, the formula II DN encoding the BanI restriction endonuclease represented by
A sequence and formula IV The DNA sequence encoding the BanI methylase represented by was determined.

The N-terminal amino acid sequence determined from the nucleotide sequence is the purified Ba
It coincided with the N-terminal amino acid sequence obtained from the nI restriction endonuclease, BanI methylase preparation. Here, it becomes possible to collect each gene using the DNA sequence whose nucleotide sequence has been determined, and at the same time, using the method of gene manipulation, Ba
A production system for nI restriction endonuclease, BanI methylase can be constructed. Any of the systems using Escherichia coli, yeast, or animal cells may be used to carry out the substance production using the method of genetic manipulation. ("Biotechnology Research Method II", pp126-150, pp170-204 (1986), edited by the Japanese Biochemical Society). For example, in order to produce BanI restriction endonuclease using E. coli as a host, a control site for expression in the above DNA sequence, for example, a DNA sequence in which a promoter of the tryptophan operon and an SD sequence are added, and a vector DNA such as pBR322 in which a BanI methylase gene is added. And then introduced into E. coli.

When yeast is used as a host, the inhibitory acid phosphatase gene and the promoter of the glyceroaldehyde 3-phosphate dehydrogenase gene are known as the expression control site, and the replication origin in yeast is 2 μm.
It is preferable to use those derived from m plasmid or yeast chromosome. When using an animal cell as a host, SV used as an expression control site
Since the 40 promoter and the promoter of the HB virus gene are known, it is preferable to use SV40 or polyoma virus as the replication initiation. Further, when using only the DNA encoding the BanI restriction endonuclease, the host may not be able to grow, so that the DNA encoding the BanI restriction endonuclease of the present invention is used in combination with the DNA encoding the BanI methylase described above. By constructing such an expression system, BanI restriction endonuclease can be produced in large quantities. When it is desired to produce only BanI methylase, this enzyme alone can be produced in large quantities by using only the BanI methylase gene in the above expression system.

 The present invention will be specifically described below with reference to examples.

Example 1 (1) Preparation of chromosomal DNA containing BanI restriction endonuclease gene Bacillus anoillinolyticus (Bacillusa neurino)
lyticus) IAM1077 was inoculated into 50 ml of L-broth medium (10 g of tryptone (Difco), 5 g of yeast extract, 10 g of NaCl adjusted to pH 7.0 per 1 pure water), and shake culture was performed at 30 ° C. for 14 hours. The post-microbial cells were collected. Next, collect 10 mg / m
l lysozyme (manufactured by Taiyo Kagaku Co., Ltd.), 20% sucrose, 1 mM
The cells were suspended in 20 ml of 50 mM Tris-HCl buffer (pH 7.6) containing EDTA and left standing at 37 ° C for 10 minutes. Next, 44 ml of 0.1 M EDTA solution (pH 9.6) containing 1% lauroyl sarcosinic acid and 5.44 mg / m
2.0 ml of the pronase solution (1) was added, and the mixture was allowed to stand at 500 ° C for 30 minutes. Next, 66 g of cesium chloride and 3.3 ml of an ethidium bromide solution containing 10 mg / ml were added, mixed, and then centrifuged at 38,000 rpm for 40 hours. Remove the DNA with a syringe, n-
Ethidium bromide was removed by butanol extraction and dialyzed against 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA to obtain about 500 μg of chromosomal DNA.

(2) Insertion of a chromosomal DNA fragment into a vector About 3 μg of the chromosomal DNA obtained in (1), 0.02 unit of SAu3AI restriction endonuclease was added, and the reaction was carried out at 37 ° C. for 1 hour to partially decompose it. Next, 4 μl of BamHI restriction enzyme was added to 1 μg of vector plasmid pBR322 (manufactured by Toyobo Co., Ltd.), and this was completely decomposed by carrying out a reaction at 37 ° C. for 1 hour. By carrying out the reaction for 1 hour, the phosphoric acid at the 5'end was removed.
3 μg of chromosomal DNA fragment obtained by the above method and 1
Chromosomal DNA was integrated by mixing μg of pBR322 DNA fragment and further ligating 5 units of T4 phage-derived DNA ligase in the presence of 1 mM ATP and 5 mM dithiothreitol at 15 ° C for 16 hours. The plasmid DNA was obtained.

(3) Transformation with recombinant plasmid containing BanI restriction endonuclease gene Escherichia coli HB101 strain which is a hybrid strain of Escherichia coli k-12 strain and Escherichia coli B strain L
Inoculate 10 ml of B medium (10 g of tryptone (Difco), 5 g of yeast extract, 10 g of NaCl adjusted to pH 7.0 per 1 pure water),
The cells were harvested by shaking culture at 37 ° C. and growing to the logarithmic growth phase. This was suspended in a solution of 0.03M CaCl ₂ at a final concentration under ice cooling to obtain competent cells. The lysis solution of the plasmid DNA obtained in (2) was added to this cell suspension, and the mixture was allowed to react for 60 minutes under ice-cooling and subjected to heat shock at 42 ° C for 1-2 minutes to take the plasmid DNA into the cells. I put it in. Then, this cell suspension was separately inoculated into the LB medium, shake-cultured at 37 ° C. for 3-5 hours, and a strain producing a BanI restriction endonuclease was isolated, and Escherichia coli HB101 (pBanIRM8) (FERM P -9424) was obtained. The restriction enzyme map of the plasmid pBanIRM8 contained in this transformed microorganism is shown in FIG. Further, pBanIRM8 was subcloned to isolate a strain producing BanI methylase to obtain Escherichia coli (pBanIM4) (FERM P-10741).

(4) Ban by Escherichia coli HB101 (pBanIRM8)
Production of I restriction endonuclease (3) Transformant Escherichia coli HB10
1 (pBanIRM8) (FERM P-9424) was subjected to aeration and stirring culture at 37 ° C. for 16 hours in the 200 L fermentation tank of the LB medium. This was centrifuged to collect microbial cells and obtain 385 g of cultured bacterial cells. Obtained fungus body 10
BanI restriction endonuclease was purified from 0 g. Fungus body 10
0 g was suspended in 500 ml of Buffer A (20 mM Tris-HCl buffer pH 7.5 containing 10 mM MgCl ₂ , 7 mM _2- mercaptoethanol). This suspension was crushed with an ultrasonic crusher sonicator for 10 minutes, and then a supernatant was prepared. Add polyethyleneimine to 0.2% in the presence of 0.2M NaCl, and add 8000r.
After centrifugation at pm for 20 minutes, a supernatant was obtained. Ammonium sulfate was added to this supernatant liquid to 70% saturation, and the precipitate was collected after precipitation with ammonium sulfate.
The suspension was suspended in 20 mM Tris-HCl buffer (pH 7.5) containing Cl ₂ , 7 mM _2- mercaptoethanol and dialyzed. After dialysis, phosphocellulose column (volume 200 ml) chromatography was performed. BanI restriction endonuclease has KCI concentration
It was eluted around 0.5M. The eluted enzyme solution was put into a dialysis tube and dialyzed. The dialysate was further subjected to hydroxyapatite column (80 ml) chromatography. Phosphoric acid concentration
The BanI fraction was eluted at 0.3M. After dialysis of this fraction
DEAE-Sepharose (40 ml) chromatography
BanI was found to elute in the washed fraction. After this fraction was concentrated, Sephacryl S-200 gel filtration was performed. The gel filtered BanI fraction was placed in a dialysis tube and dialyzed against a phosphate buffer containing 2 mM mercaptoethanol and 50% glycerol to obtain 2.8 ml of enzyme solution.

When this purified BanI enzyme was examined for protein purity by SDS-PAGE, it was found to be a single BanI protein. The molecular weight of BanI was about 72,00 depending on the elution position of Sephacryl S-200.
It was estimated to be 0 (dimer with a subunit molecular weight of 36,000 by SDS-PAGE) (Fig. 2). When the N-terminal amino acid sequence of this enzyme preparation was determined using an amino acid sequencer, Al
a-Gln-Leu-Lys-Tyr-Asn-Lys-Asp-Ile-Asp-Gl
It was found to be u-Leu-Glu.

(5) Transformation with recombinant plasmid containing BanI methylase gene Recombinant plasmid pBanIRM8 containing DNA of BanI restriction endonuclease BanIII containing only methylase gene
-SphI fragment was inserted into the AccI-SphI site of pUC18 and pBanIM4
Was prepared (Fig. 3). Then, E. coli HB101 was transformed with pBanIM4 by a conventional method.

(6) BanI by Escherichia coli HB101 (pBanIM4)
Production of methylase E. coli HB101 (pBanIM4) (FERM P-10741) containing pBanIM4 having DNA encoding BanI methylase was transformed into L-
The cells were cultured in a broth medium using a 0.5 l flask to collect the cells. Suspend the cells in 50 ml of Buffer A and sonicate (3A, 10
Minutes) The supernatant was obtained by centrifugation at 35,000 rpm for 30 minutes. The supernatant was subjected to phosphocellulose column (40 ml) chromatography. Since BanI methylase passes through the column, it was collected and subjected to DEAE column chromatography. BanI methylase is eluted with KCl 0.3M. This fraction was again subjected to phosphocellulose column (20 ml) chromatography. BanI methylase was eluted with 0.45 M KCl. This fraction was concentrated and subjected to Sephacryl S-200 gel filtration (2 cm × 37 cm). The BanI methylase fraction was dialyzed and further subjected to heparin (10 ml) column chromatography. Concentrate the eluted fraction with polyethylene glycol, then 50%
It was dialyzed against a glycerol solution. 4 kg of total activity
We obtained BanI methylase.

When this bleached product was examined for protein purity by SDS-PAGE and then silver staining, a single band (Fig. 8) was shown. In this case, the molecular weight was about 44,000. It was about 45,000 by the gel filtration method (Fig. 4). This enzyme was presumed to be a monomer enzyme.

(7) Isolation of DNA encoding BanI restriction endonuclease and BanI methylase and determination of nucleotide sequence Escherichia coli HB101 (pBanIRM8) (FERM P) producing the BanI restriction endonuclease obtained in (3)
-9424) and the recombinant plasmid (pBan
IRM8) was prepared. Prepared pBanIRM8 with restriction enzyme HindII
After digesting with I and SphI, the DNA fragment (6.2 kb) encoding BanI restriction endonuclease and BanI methylase was excised and blunt-ended into Klenow fragment.
Inserted into the incII site and created pBanIRM6.2 (5th
Figure). A restriction enzyme map of the pBanIRM8-derived DNA fragment inserted into pUC18 is shown (Fig. 6).

The BanI restriction endonuclease and BanI methylase coding regions in this insert are shown in FIG. In the figure, BanIR represents the coding region of BanI restriction endonuclease, and BanIM represents the coding region of BanI methylase. Cmp1 to Cmp4 are custom mate primers used for determining the nucleotide sequence.

Next, pBanIRM6.2 was digested with restriction enzymes BamHI and KpnI to obtain BamH.
An I-KpnI DNA fragment was prepared. This DNA fragment was subjected to gradual deletion (Deletion) from the BanI restriction endonuclease gene side with exonuclease III and mung bean nuclease, and the resulting gradual deletion mutant was prepared and sequenced by the dioxy method. . Thereby, the base sequence of one strand of DNA was determined. Regarding the other strand, nucleotides (17mer) complementary to the base sequence determined as described above were synthesized, and the base sequence was determined by the dioxy method using these as primers. As a result, the nucleotide sequences of the DNA encoding the BanI restriction endonuclease and the DNA encoding the BanI methylase represented by the above formulas II and IV were determined.

In addition, the amino acid sequences of the above formula I corresponding to the base sequences of these II and IV and the formula III At the N-terminus matched the amino acid sequences at the N-terminus obtained from the purified BanI restriction endonuclease and BanI methylase preparations, respectively.

(Effect of the invention) The present invention has made it possible to provide a DNA encoding a BanI restriction endonuclease. This makes it possible to efficiently produce a large amount of a pure BanI restriction endonuclease preparation containing no contaminating enzyme efficiently and in large amounts by using a gene manipulation technique.

[Brief description of drawings]

Fig. 1 shows the restriction enzyme map of pBanIRM8 plasmid, Fig. 2 shows the measurement results of the molecular weight of pBanI restriction endonuclease, Fig. 3 shows the construction diagram of pBanIRM8 to pBanIM4, and Fig. 4 shows the measurement of BanI methylase molecular weight. Figure showing the results, No. 5
The figure is a construction diagram of pBanIRM6.2, Fig. 6 is a restriction map of the pBanIRM8-derived DNA fragment inserted into pUC18 when constructing pBanIRM6.2, and Fig. 7 is a pBanIRM8-derived DNA fragment inserted into pUC18. BanI restriction endonuclease and Ban in Escherichia coli
Coding region of I-methylase, and D of these enzymes
It is a figure which shows the strategy of NA base sequence determination. FIG. 8 is a photograph showing the electrophoretic pattern of BanI methylase obtained by the present invention.

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Claims (2)

[Claims] 1. A Bacillus anoillinolyticus (Bacillus)
aneurinolyticus) -derived BanI restriction endonuclease, which has a nucleotide sequence encoding the amino acid sequence represented by the following formula I. Formula I 2. An expression vector containing the DNA fragment according to claim 1.