JPH0630576B2 - Method for producing Ban I restriction endonuclease - Google Patents

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a microorganism transformed with a new recombinant plasmid comprising a chromosomal DNA fragment incorporating the gene for BanI restriction endonuclease, and a method for producing BanI restriction endonuclease from said microorganism.

(PRIOR ART) Type II restriction enzymes are extremely specific enzymes that recognize and cleave specific base sequences in deoxyribonucleic acid (DNA) chains, and due to this excellent specificity, they are widely used in the field of genetic engineering.

To date, about 100 types of type II restriction enzymes have been discovered from bacteria and the like and have been commercialized.
anI is one of the type II restriction enzymes, and is an enzyme that recognizes and cleaves GGPyPuCC in the DNA base sequence.
It is known that acetylcholine is produced in the yeast Lactobacillus Aneurinolyticus IAM1077 [Nucleic Acids Research 10,
5747(1982)].

To utilize type II restriction enzymes in the field of genetic engineering, the following four conditions must be satisfied at a minimum:

That is, it does not contain any other restriction enzymes.

Contains no phosphatase.

Does not contain non-specific DNase.

It is 3' and 5' exonuclease free.

Therefore, commercially available restriction enzymes are used for nucleic acid removal,
It is highly purified by combining salting out, gel filtration, ion exchange chromatography, affinity chromatography, etc. The present inventors have also tried the same method for BanI as for other restriction enzymes.
recognizes ATCGAT in addition to BanI, and recognizes the restriction enzymes BanIII and GPuGCPyC which cleave it;
Since the restriction enzyme BanII, which cleaves this, is also produced at the same time, it is very difficult to remove this.

(Object of the Invention) The present inventors have solved the problems of the above-mentioned method, namely BanIII and Ba
In order to overcome the problem of simultaneously producing nII and BanI, we have conducted extensive research to develop a strain that produces only BanI.
The inventors have succeeded in extracting a chromosomal DNA fragment containing the gene for BanI restriction endonuclease from Bacillus neurinolyticus IAM1077, incorporating the fragment into a vector to prepare a recombinant plasmid, and obtaining a microorganism transformed by introducing the plasmid.
The present invention was completed based on this new finding.

(Configuration of the Invention) That is, the present invention relates to a method for producing Bacillus anenolyticus (Baci
BanI derived from Illus aneurinolyticus IAM1077
Restriction enzymes, including restriction endonuclease genes,
Chromosomal DNA of 3.6 kb cleaved by u3AI
The present invention relates to a microorganism belonging to the genus Escherichia transformed with a recombinant plasmid capable of replicating in Escherichia coli containing A, and a method for producing BanI restriction endonuclease, which comprises culturing the transformed microorganism and recovering BanI restriction endonuclease from the culture.

Since the transformant strain into which the above-mentioned recombinant plasmid of the present invention has been introduced produces only BanI restriction endonuclease, it is not necessary to remove BanIII and BanII restriction endonucleases in the purification process, and it has become possible to easily produce large amounts of BanI restriction endonuclease.

The present invention will be described in detail below.

(a) Plasmids and their Preparation The novel plasmids of the present invention can be prepared, for example, from Escherichia coli (Esch).
The colicin E1 element, known as an extrachromosomal gene (plasmid) of microorganisms belonging to the genus Bacillus aneurinolyticus, is a plasmid that can grow in cultured cells.
The vector DNA may be a DNA fragment extracted from a naturally occurring vector or a DNA fragment lacking a portion of the DNA other than the portion essential for proliferation. For example, the vector DNA may be a DNA fragment extracted from a naturally _occurring vector or a DNA fragment lacking a portion of the DNA other than the portion essential for proliferation.
Examples of such vectors include MB9 strains, pBR322 strains, pSC101 strains, R6K strains, and lambda phage strains.

The chromosomal DNA fragment may be integrated into the vector DNA by any known method, for example, by treating the chromosomal DNA with an appropriate restriction enzyme (endonuclease) to cleave the chromosomal DNA at a specific site, mixing it with a similarly treated vector DNA, and religating the resulting DNA with a ligase.

The vector DNA used was pBR322 plasmid, which was transformed with Bacillus anenolyticus (
The novel plasmid p
The restriction enzyme map of pBAN11 plasmid is shown in FIG. 1. In FIG. 1, the area shown in a white frame indicates a chromosomal DNA fragment containing the BanI restriction endonuclease gene. As is clear from FIG. 1, this plasmid contains the Bacillus aneurinolyticus IAM107 gene at the BamHI site of the restriction enzyme site of the pBR322 plasmid.
7 containing the BanI restriction endonuclease gene
The A fragment is a circular molecule having an integrated length of 8 Kb of base pairs.

(b) Preparation of Microorganisms The composites of the chromosomal DNA fragment and the vector DNA thus obtained are introduced into recipient microbial cells by a known transformation method, for example, the shot gun method, to obtain a transformant having both the desired genetic traits and the traits of the vector DNA.

The recipient bacteria was the aforementioned Escherichia coli HB10.
Microorganisms that are usually used in this technical field, such as Escherichia coli C600, DP, supF, x1776, and LE392, can be advantageously used. A typical example of such a microorganism is the Escherichia coli HB101 strain [Molecular.
Cloning A Laboratory Manual, (Mo
See Lecular Cloning Laboratory Manual p.504 (1982); genotype ^F- , hsd s20 ( _r8 , _M8 ) rec A13, ara-14, pro
A2, lacY1, gal K2, rps L20(Sm′), xy1-5, mtl-1, sup E44
λ ⁻ )].

The microorganism obtained by transformation of the Escherichia coli HB101 strain with the plasmid pBAN11 is a novel microorganism.
01 (pBAN11) [Echerichia coli HB101
(pBAN11)] and was deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology on June 18, 1987, under the deposit number Fermentation Research Institute Deposit No. 9424 (FERM P-
The bacteriological properties of the thus obtained Escherichia coli HB101 (pBNA11) were as follows:
Comparing the properties of the DNA recipient Escherichia coli HB101 strain, the two are identical except that the former produces BanI restriction endonuclease and has ampicillin resistance, whereas the latter does not have these properties.

(c) Production of restriction endonuclease To culture the transformant obtained in step (b), a medium suitable for the production of a substance generated by a specific genetic information and suitable for the growth of the host microorganism can be used. In the method of the present invention, a medium such as LB medium (tryptone, yeast extract, salt), BPB medium (Difco; polypeptone, yeast extract, potassium phosphate), or nutrient agar medium (Difco 0000; polypeptone, yeast extract, potassium phosphate), which is generally used as a growth medium for Escherichia coli, can be used.
1) Tryptone/sodium chloride medium or the like can be used as the base medium.

In addition, amino acids, carbon sources, nitrogen sources, etc., may be added as necessary.
Nutrients such as vitamins may also be added.

The culture method may be carried out under conditions such as pH, temperature, and oxygen supply that are suitable for the growth of Escherichia microorganisms. It is preferable to grow the microorganism until the amount of the microorganism reaches a maximum after inoculation into the medium, i.e., until the late logarithmic growth stage.
The temperature is preferably up to 37° C. and the pH condition is preferably in the range of 5 to 8, particularly preferably near neutral.

The resulting cells are harvested and extracted by centrifugation, ultrasonic disruption, or the like, and then the BanI restriction endonuclease can be obtained by a combination of nucleic acid removal methods, salting out, gel filtration, ion exchange chromatography, affinity chromatography, and the like.

(Effects of the Invention) The plasmid of the present invention and the transformed strain into which the plasmid is introduced are B
Since only the BanI restriction endonuclease is produced, there is no need to remove the BanIII and BanII restriction endonucleases during the purification process, which makes it possible to easily produce large amounts of the BanII restriction endonuclease.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these in any way.

Example 1. (1) Preparation of chromosomal DNA carrying the BanI restriction endonuclease gene
yticus IAM1077 was cultured in L-broth medium [10 g tryptone (Difco), 5 g yeast extract, and 10 g N
aCl (10 g, pH adjusted to 7.0) was inoculated into 50 ml of the medium and cultured with shaking at 30° C. After 14 hours, the cells were harvested. The harvested cells were then diluted with 10 mg/ml lysozyme (Taiyo Kagaku Co., Ltd.), 20% sucrose, 1 mM EDTA,
20 mM Tris-HCl buffer (pH 7.6) containing TA
The mixture was then suspended in 0.1M EDTA solution (pH 6.0) containing 1% lauroyl sarcosinic acid and allowed to stand at 37° C. for 10 minutes.
9.6) 2.0 ml of 44 mg/ml and 5.44 mg/ml pronase solutions were added and allowed to stand at 50° C. for 30 minutes. Next, 66 g of cesium chloride and 3.3 ml of 10 mg/ml ethidium bromide solution were added and mixed, followed by 38.0 ml of ethidium bromide solution.
The DNA layer was extracted with a syringe, and the ethidium bromide was removed by extraction with n-butanol.
The mixture was dialyzed against 0 mM Tris-HCl buffer (pH 8.0) to obtain about 500 μg of chromosomal DNA.

(2) Insertion of a chromosomal DNA fragment into a vector Add 0.02 units of Sau3AI restriction endonuclease to 3 μg of the chromosomal DNA obtained in (1)
This was partially decomposed by reacting at 37° C. for 1 hour. Next, 4 units of BamHI were digested for 1 μg of vector plasmid pBR322 [manufactured by Toyobo Co., Ltd.].
This was completely decomposed by adding a restriction endonuclease and reacting at 37° C. for 1 hour, and one unit of alkaline phosphatase was further added and reacted at 37° C. for 1 hour to remove the phosphate at the 5' end.
The 3 μg chromosomal DNA fragment obtained by the above method and 1 μg of pBR322 DNA fragment were mixed and further
5 mM ATP and 5 mM dithiothreitol
A ligation reaction was carried out at 15° C. for 16 hours using a DNA ligase derived from _T4 phage, thereby obtaining a plasmid DNA incorporating the chromosomal DNA.

(3) Transformation of E. coli K-12 and E. coli B with recombinant plasmids containing the BanI restriction endonuclease gene
A hybrid strain of Escherichia coli HB10
One strain was cultured in LB medium [1 part tryptone (Difco) per 1 part of purified water].
The cells were inoculated into 10 ml of a mixture of 10 g of maltodextrin, 5 g of yeast extract, and 10 g of NaCl, adjusted to pH 7.0, and cultured with shaking at 37°C until they reached the logarithmic growth phase, after which they were harvested. The cells were suspended in a solution with a final concentration of 0.03 M _CaCl2 under ice cooling to obtain competent cells. The solution of the plasmid DNA obtained in (2) was added to the cell suspension, reacted for 60 minutes under ice cooling, and then heat-shocked at 42°C for 1-2 minutes to incorporate the plasmid DNA into the cells. The cell suspension was then inoculated into the LB medium separately and cultured with shaking at 37°C for 3-5 hours to carry out a transformation reaction, after which a strain having ampicillin resistance and producing BanI restriction endonuclease was isolated and transformed into Escherichia coli HB101 (pBAN11) (Microbial Strain No. 9).
The compound was obtained as No. 424.

(4) Production of BanI restriction endonuclease by Escherichia coli HB101 (pBAN11)
1 (pBAN11) (Microbial Strain No. 9424) was incubated at 37° C. in a 2-volume flask containing 500 ml of the LB medium.
The culture was shaken for 16 hours. The cells were collected by centrifugation, washed, suspended in 25 ml of 20 mM Tris-HCl buffer (pH 7.5) containing 10 mM _MgCl2 and 7 mM 2-mercaptoethanol, and subjected to ultrasonic disruption at 0°C for 10 minutes. The enzyme extract was obtained by centrifugation at 12,000 rpm for 10 minutes. Ammonium sulfate powder was then added to the enzyme extract under ice cooling to dissolve it, and the 30-80% saturated fraction (saturation expressed by Osborne method) was collected by centrifugation. The collected precipitate was dissolved in 2 ml of 10 mM phosphate buffer (pH 7.5) containing 2 mM mercaptoethanol and 5% glycerol, and further placed in a dialysis tube and dialyzed overnight against 100 times the amount of the same buffer. The column was then adsorbed onto a phosphocellulose (Whatman) column (volume 20 m) equilibrated with the same buffer. After washing with 5 volumes of the same buffer, elution was performed with a 0-1.0M KCl gradient.
The enzyme solution was eluted at a Cl concentration of about 0.5 M. The eluted enzyme solution was placed in a dialysis tube and diluted with 2 mM mercaptoethanol, 5 mM
The enzyme solution was dialyzed against phosphate buffer containing 0.01% glycerol to obtain 0.2 ml of the enzyme solution, the enzyme activity of which was measured and found to be 10,000 units.

The enzyme solution obtained in this manner does not contain other restriction endonucleases, phosphatases, non-specific DNases, etc., and can be used in the field of genetic engineering. The activity of BanI restriction endonuclease is measured by dissolving 1 μg of λ-DNA in 45 μl of reaction solution consisting of 20 mM Tris-HCl buffer, 10 mM magnesium chloride solution, 50 mM ammonium sulfate, and 7 mM 2-mercaptoethanol, adding 5 μl of enzyme solution to the mixture, reacting at 37° C. for 1 hour, and then performing agarose electrophoresis. One unit of enzyme activity is 1 μl per hour at 37° C. and pH 7.5.
It refers to the enzyme activity that completely degrades γ-DNA of g.

[Brief description of the drawings]

FIG. 1 shows the restriction enzyme map of the pBAN11 plasmid of the present invention.

Claims (1)

[Claims] Claim 1: Bacillus agonistinolyticus
BanI derived from U.s. aneurinolyticus IAM 1077
Restriction enzymes, including restriction endonuclease genes,
Chromosomal DNA of 3.6 kb cleaved by u3AI
Escherichia transformed with a recombinant plasmid capable of replicating in E. coli, which incorporates the A fragment.
The method comprises culturing a microorganism belonging to the genus BanI and extracting BanI restriction endonuclease from the culture.
Methods for producing restriction endonucleases.