JPH0630577B2 - Ban III-Method for producing restriction endonuclease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is a microorganism transformed by introducing a new recombinant plasmid incorporating a chromosomal DNA fragment containing a gene for BanIII restriction endonuclease, and BanIII restriction from the microorganism. It relates to a method for producing an endonuclease.

(Prior Art) A 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 due to its excellent specificity, it is used in the field of genetic engineering. Widely used in.

To date, about 100 types of type II restriction enzymes have been discovered from bacteria and commercialized. B according to the invention
anIII is also one of the type II restriction enzymes, and DNA
Bacillus anoillinolyticus (Bacill) is an enzyme that recognizes and cleaves ATCGAT in the nucleotide sequence of
us Aneurinolyticus) is known to be produced in IAM1077 [Nucleic Acids Research 10, 57
47 (1982)].

In order to use the type II restriction enzyme in the field of genetic engineering, at least the following four conditions must be satisfied.

That is, it does not contain other restriction enzymes.

Does not contain phosphatase.

Does not contain non-specific DNase.

Free of 3'and 5'-exonucleases.

Therefore, the commercially available restriction enzymes are the nucleic acid removal method,
It is highly purified by a combination of salting out method, gel filtration method, ion exchange chromatography method, affinity chromatography method and the like. The present inventors have tried a method similar to other restriction enzymes for BanIII, but particularly for BanIII, Bacillus aneurinolyticus IAM10
77 recognizes GGPyPuCC in addition to BanIII, recognizes the restriction enzymes BanI and GPuGCPyC that cut it, and simultaneously produces the restriction enzyme BanII that cuts it. Therefore, it was very difficult to remove it.

OBJECTS OF THE INVENTION The present inventors have found that the drawbacks of the above methods are BanIII and Ba.
The intent of this study was to solve the problem of producing nII at the same time and to create a strain producing only BanI. As a result, the Bacillus anoillinolyticus (Bacillus
neurinolyticus) IAM1077, a chromosomal DNA fragment containing a BanIII restriction endonuclease gene is extracted, a recombinant plasmid is prepared by incorporating this into a vector, and a transformed microorganism is obtained by introducing the plasmid, and The microorganism is BanII
Discovered the fact that it only produces I. The present invention has been completed based on this new finding.

(Structure of the Invention) That is, the present invention relates to Bacillus anoylinolyticus (Baci
llus aneurinolyticus) BanII derived from IAM1077
Restriction enzymes, including the I restriction endonuclease gene, S
Chromosome D, which is 7.6 kb and has been cleaved by au3AI
Characterized by culturing a microorganism belonging to the genus Escherichia transformed with a recombinant plasmid capable of replicating in Escherichia coli incorporating NA and a transformed microorganism, and collecting a BanIII restriction endonuclease from the culture. It relates to a method for producing BanIII restriction endonuclease.

Since the transformant introduced with the above recombinant plasmid of the present invention produces only BanIII restriction endonuclease, it is not necessary to remove BanI restriction endonuclease and BanII restriction endonuclease in the purification step, and a large amount of BanIII restriction endonuclease can be obtained. It became possible to manufacture easily.

The present invention will be described in detail below.

(A) Plasmid and its preparation The novel plasmid of the present invention is, for example, Escherichia (Esch.
Escherichia coli (E. coli) known as an extrachromosomal gene (plasmid) of a microorganism belonging to the genus erichia) and a plasmid having a form capable of proliferating in a cultured cell, Bacillus aneurinolyticus (Bacillus aneurinolyticus)
A plasmid incorporating a DNA fragment cleaved by a restriction enzyme, Sau3AI, containing a BanIII restriction endonuclease gene derived from IAM1077, and the above-mentioned vector DNA is essential for proliferation in addition to those extracted in nature. A part of DNA other than the part may be deleted, for example, ColE ₁ strain,
Examples include the pMB9 line, the pBR322 line, the pSC101 line, the R6K line, and the Lambdarge line.

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 (Endonuclease), then mixed with similarly treated vector DNA, and religated with ligase.

As the vector DNA, pBR322 plasmid was used, to which Bacillus anoillinolyticus (Bacillus
aneurinolyticus) IAM1077, a new plasmid p
BAN31 is obtained. A restriction map of the pBAN31 plasmid is shown in FIG. In FIG. 1, a white box indicates a chromosomal DNA fragment containing a BanIII restriction endonuclease gene. As is clear from FIG. 1, this plasmid was found at the BamHI site of the restriction enzyme site of the pBR322 plasmid, at the Bacillus aneurinolyticus IAM107.
D containing 7 BanIII restriction endonuclease genes
It is a circular molecule having a 12 Kb base pair with an incorporated NA fragment.

(B) Preparation of microorganism The thus obtained chromosomal DNA fragment-vector DNA combination is introduced into a microbial cell of a recipient bacterium by a known transformation method, for example, the shot gun method. Then, a transformant having both the desired genetic trait and the vector DNA trait is obtained.

As the recipient bacterium, the above-mentioned Escherichia coli HB10
Microorganisms usually used in this type of technical field, such as 1, C600, DP, supF, χ1776, LE392, etc. are advantageously used. A typical example thereof is the Escherichia coli HB101 strain [Molecular
Cloning A Laboratory Manual, (Mo
See lecular Cloning Laboratory Manual P.504 (1982); Genetic trait F ⁻ , hsd s20 (r ₈ , M ₈ ) rec A13, ara-14, pro.
A2, lacY1, gal K2, rps L20 (Sm '), xy1-5, mtl-1, sup E44
λ ⁻ )].

A microorganism obtained by the transformation method by introducing the above-mentioned plasmid pBAN31 into this Escherichia coli HB101 strain is a novel microorganism, and Escherichia coli HB1.
01 (pBAN31) [Echerichia coli HB101
(PBAN31)] and deposited on June 18, 1987 at the Institute for Microbial Technology, Institute of Industrial Science, the deposit number is Micromachine Research Institute No. 9423 (FERM P-
9423). The mycological properties of Escherichia coli HB101 (pBNA31) thus obtained were
Compared with the properties of the Escherichia coli HB101 strain that is a DNA recipient, while the former has production of BanIII restriction endonuclease and ampicillin resistance,
The latter is exactly the same except that the latter does not have these properties.

(C) Production of restriction endonuclease In order to culture the transformant obtained in step (b), it is a medium suitable for the production of substances produced by specific genetic information and suitable for the growth of host microorganisms. In the method of the present invention, LB medium (tryptone, yeast extract, salt), BPB medium (Difco; polypeptone, yeast extract, potassium phosphate), which is usually used as a growth medium for Escherichia coli, and nutrients can be used.・ Agar medium (Difco000
1), tryptone / saline medium, etc. prepared as a basic medium may be used.

In addition to the carbon and nitrogen sources, amino acids,
Nutrients such as vitamins may be added.

The culture method, pH, temperature, it is possible to adopt conditions suitable for the growth of microorganisms of the genus Escherichia genus as conditions such as oxygen supply, after inoculating the microorganism into a medium, the microorganism grows and It is preferable to grow when the amount reaches the maximum, that is, until late logarithmic growth. The culture temperature is usually 30
It is suitable that the pH condition is ˜37 ° C. and the pH range is 5˜8, especially around neutral.

After collecting the obtained bacterial cells, centrifugation, extraction by ultrasonication, etc., and then combining nucleic acid removal method, salting out method, gel filtration method, ion exchange chromatography method, affinity chromatography method, etc. BanIII restriction endonuclease can be obtained.

(Effect of the Invention) The plasmid of the present invention and the transformant into which it is introduced are B
to produce only anIII restriction endonuclease,
It was not necessary to remove the BanI and BanII restriction endonucleases in the purification step, which made it possible to easily produce a large amount of BanIII restriction endonuclease.

(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1. (1) Preparation of chromosomal DNA containing BanIII restriction endonuclease gene Bacillus aneurinol
yticus) IAM1077 in L-broth medium [10 g of tryptone (Difco) per 1 pure water, 5 g of yeast extract, N
10 g of aCl adjusted to pH 7.0] was inoculated into 50 m, and shake culture was carried out at 30 ° C. After 14 hours, the bacterial cells were collected. Next, the collected bacterial cells were 10 mg / m of lysozyme [manufactured by Taiyo Kagaku Co., Ltd.], 20% sucrose, 1 mM ED.
50 mM Tris-HCl buffer (pH 7.6) containing TA 20
m and suspended at 37 ° C. for 10 minutes. Next, 0.1 M EDTA solution containing 1% lauroyl sarcosinic acid (pH
9.6) 44 m and 2.04 m of a 5.44 mg / m pronase solution were added, and the mixture was allowed to stand at 50 ° C for 30 minutes. Next, 66 g of cesium chloride and 3.3 m of an ethidium bromide solution of 10 mg / m were added, and after mixing, 38.0
Centrifugation was performed at 00 rpm for 40 hours. The DNA layer was extracted with a syringe, ethidium bromide was removed by n-butanol extraction, and 1 mM EDTA was added.
About 500 μg of chromosomal DNA was obtained by dialysis against 0 mM Tris-HCl buffer (pH 8.0).

(2) Insertion of chromosomal DNA fragment into vector Add 0.02 units of Sau3AI restriction endonuclease to 3 μg of chromosomal DNA obtained in (1),
This was partially decomposed by carrying out a reaction at 37 ° C. for 1 hour. Next, 4 units of BamHI for 1 μg of the vector plasmid pBR322 [manufactured by Toyobo Co., Ltd.]
This is completely decomposed by adding a restriction endonuclease and performing a reaction at 37 ° C. for 1 hour, and further adding 1 unit of alkaline phosphatase and performing a reaction at 37 ° C. for 1 hour, whereby the 5'terminal phosphorus is The acid was removed.
3 μg of the chromosomal DNA fragment obtained by the above method and 1 μg of the pBR322 DNA fragment were mixed, and further mixed for 1 m.
5 in the presence of MATP and 5 mM dithiothreitol
A plasmid DNA incorporating chromosomal DNA was obtained by carrying out a ligation reaction at 15 ° C. for 16 hours using a DNA ligase derived from a unit of T ₄ phage.

(3) Transformation with recombinant plasmid containing BanIII restriction endonuclease gene Escherichia coli K-12 strain and Escherichia coli B
Hybrid strain of Escherichia coli HB10
1 strain in LB medium [tryptone per 1 pure water (Difco)
10 g, yeast extract 5 g, and NaCl 10 g adjusted to pH 7.0] were inoculated into 10 m, cultured at 37 ° C. with shaking, grown to a logarithmic growth phase, and then collected. 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 reacted for 60 minutes under ice-cooling and heat shocked at 42 ° C for 1 to 2 minutes to bring the plasmid DNA into the cells. I took it in. Then, this cell suspension was separately inoculated into the LB medium, shake-cultured at 37 ° C. for 3 to 5 hours to carry out a transformation reaction, and then a strain having ampicillin resistance and producing a BanIII restriction endonuclease was obtained. Isolation was performed to obtain Escherichia coli HB101 (pBAN31) (Ministry of Industrial Science, No. 9423).

(4) Production of BanIII restriction endonuclease by Escherichia coli HB101 (pBAN31) (3) Transformant Escherichia coli HB10 obtained in (3)
1 (pBAN31) (Microtechnology Research Institute, No. 9423) in a 2 volume flask containing 500 m of the LB medium at 37 ° C.
Shaking culture was performed for 16 hours. After centrifuging, the cells were collected, washed and suspended in 25m of 20mM Tris-HCl buffer (pH 7.5) containing 10mM MgCl ₂ , 7mM 2-mercaptoethanol, and ultrasonically disrupted at 0 ° C for 10 minutes. It was Further, the enzyme extract was obtained by centrifugation at 12,000 rpm for 10 minutes. Next, ammonium sulfate powder was added to and dissolved in this enzyme extract under ice-cooling, and a 30-80% saturated fraction (the degree of saturation is indicated by the Osborne method) was collected by centrifugation. The recovered precipitate was dissolved in 2m of 10mM phosphate buffer (pH 7.5) containing 2mM mercaptoethanol and 5% glycerol, put into a dialysis tube, and dialyzed against 100 times volume of the same buffer overnight. did. Then, it was adsorbed on a column (capacity 20 m) of phosphocellulose (manufactured by Whatman) equilibrated with the same buffer solution. After washing with 5 volumes of the same buffer, 0-1.0 MKCl gradient elution was performed. BanIII restriction endonuclease was eluted at a KCl concentration of around 0.5M. Put the eluted enzyme solution into a dialysis tube, and add 2 mM mercaptoethanol,
By dialyzing against a phosphate buffer containing 50% glycerol, 0.2 m of enzyme solution was obtained. When the enzyme activity of the obtained enzyme solution was measured, it was 10,000 units.

The enzyme solution thus obtained did not contain any other restriction endonuclease, phosphatase, non-specific DNase, etc., and could be used in the field of genetic engineering. The activity of BanIII restriction endonuclease was measured by dissolving 1 μg of λ-DNA in 45 μl of a reaction solution consisting of 20 mM Tris-hydrochloric acid buffer solution, 10 mM magnesium chloride solution, 50 mM ammonium sulfate, and 7 mM 2-mercaptoethanol. The enzyme solution is added, the reaction is carried out at 37 ° C. for 1 hour, and then the agarose is electrophoresed for measurement. One unit in enzyme activity is 1 μg per hour at 37 ° C and pH 7.5.
Enzyme activity that completely decomposes λ-DNA of

[Brief description of drawings]

FIG. 1 shows a restriction map of the plasmid of pBAN31 of the present invention.

Claims (1)

[Claims] 1. Bacillus anoylinolyticus
us aneurinolyticus) BanII derived from IAM 1077
Restriction enzymes, including the I restriction endonuclease gene, S
Chromosome D, which is 7.6 kb and has been cleaved by au3AI
Escherichia coli transformed with a recombinant plasmid incorporating the NA fragment and capable of replicating in E. coli.
a) culturing a microorganism belonging to the genus and removing BanIII from the culture.
B characterized by collecting restriction endonuclease
Method for producing anIII restriction endonuclease.