JPH0257185A - Production of novel bamhi restriction endonuclease - Google Patents

Abstract

NEW MATERIAL:A novel recombinant plasmid reproducible by Bacillus bacteria, incorporated with the chromosome DNA fragment containing BamHI restriction endonuclease gene of Bacillus amyloliquefaciens H strain origin. USE:Bacillus microorganisms are transformed with the above-mentioned plasmid to obtain a novel microorganisms capable of mass production of BamHI restriction endonuclease. PREPARATION:Plasmid such as pTB 53 as an extrachromosomal gene of Bacillus microorganisms is incorporated, by a conventional technique, with DNA fragment containing BamHI restriction endonuclease gene of Bacillus amyloliquefaciens H strain.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides a novel recombinant plasmid that incorporates a chromosomal DNA fragment containing the BamHI restriction endonuclease gene, and a method for transforming the plasmid by introducing the plasmid. The present invention relates to a novel microorganism and a novel method for producing BaIIIHI restriction endonuclease from the microorganism.

(Prior art) Type 1 restriction enzymes are highly specific enzymes that recognize and cleave specific base sequences in deoxyribonucleic acid (DNA) chains. It is widely used in

To date, approximately 100 types of type restriction enzymes have been discovered from bacteria and other sources and have been commercialized. B according to the present invention
amHI is also one of these ■ type restriction enzymes, and is one of the enzymes that are extremely frequently used in the field of genetic engineering. BamHI restriction endonuclease is a DNA
It is an enzyme that recognizes and cleaves GGATCC in the base sequence of Bacillus amyloliquefaciens (Ba
It is known that it is produced in strain H (Journ
al of Molecular Biology 9
7123 (1975), Journal of Bi
logicalchemistry 2541QO3
(1979)).

In order to use Type 2 restriction enzymes in the field of genetic engineering, it is necessary to satisfy at least the following four conditions.

In other words, ■ Contains no other restriction enzymes.

■ Contains no phosphatase.

■ Does not contain non-specific DNase.

■ Does not contain 3' and 5'-exonuclease.

Therefore, commercially available restriction enzymes are
It is purified to a high degree of purity by a combination of salting-out methods, gel filtration methods, ion exchange chromatography methods, affinity chromatography methods, etc.

However, the amount of type 1 restriction enzyme produced by microorganisms is generally small, and the amount of purified enzyme obtained is also small. In recent years, advances in genetic engineering have led to recombinant DNA technology being used for the production of many enzymes. The same holds true for type II restriction enzymes, and improvements in productivity using recombinant DNA technology have been reported for some enzymes (Jour
nal of Bacterology 16450
1 (1985), Proceedings of N.
a-eye 0nal Academy 5science 78
1503 (1981), JP-A-61-265094, JP-A-63-87982).

However, all of these reports are studies using Escherichia coli as a host, and no study using Bacillus (order Bacillus) as a host has been reported at all. Ba of the present invention
The mHI restriction endonuclease was also described in Japanese Patent Application Publication No. 13
Although there is a report in No. 87982, this study uses E. coli as a host, and is a very complicated method that uses two types of vectors.

(Object of the Invention) The present inventors worked on cloning BaIIIHI restriction endonuclease using Bacillus genus bacteria as a host, and
A chromosomal DNA fragment containing the BamHI restriction endonuclease gene was extracted from the BamHI restriction endonuclease gene (lens) H strain, and this was incorporated into a vector to create a recombinant plasmid, and by introduction of the plasmid, a transformed microorganism was successfully obtained.

Furthermore, it was discovered for the first time that in bacteria of the genus Bacillus, it is possible to incorporate genes for restriction and modification therapy into one vector. The present invention was completed based on this new knowledge.

(Structure of the Invention) That is, the present invention provides Bacillus amyloliquefaciens (B.
acillus an+ylollquefacren
s) Bacillus (B
A novel recombinant plasmid that can be replicated in bacteria of the genus Bacillus (order Baa), a new microorganism belonging to the genus Bacillus (order Baa) transformed with the plasmid, and the transformed microorganism, and BamHI restriction from the culture. BamHI characterized by collecting endonuclease
Relates to new production methods for restriction endonucleases.

The above recombinant plasmid of the present invention and the transformed strain into which it has been introduced produce BamHI restriction endonuclease in large quantities, making it possible to easily produce large quantities of 13amHI restriction endonuclease.

The present invention will be explained in detail below.

(a) Plasmid and its preparation The novel plasmid of the present invention is a plasmid that can proliferate in cultured cells, such as pTB53, which is known as an extrachromosomal gene (plasmid) of a microorganism belonging to the genus Bacillus (order Bacillus). The plasmid to be taken is Bacillus amyloliquefacins (Baallus amylollq).
This is a plasmid that incorporates a DNA fragment containing the BamHI restriction endonuclease gene derived from the BamHI restriction endonuclease gene derived from the S. uefaclens) H strain, and the vector DNA includes extracted naturally occurring DNA as well as parts of the DNA other than the parts essential for proliferation. may be partially missing.

Furthermore, any known method can be applied to incorporate the chromosomal DNA fragment into the vector DNA. For example, a method is used in which chromosomal DNA is treated with an appropriate restriction enzyme (Endonuclease) to cut it at a specific site, then mixed with similarly treated vector DNA, and religated with ligase.

Using pTB53 plasmid as vector DNA,
This is accompanied by Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
By integrating the chromosomal DNA fragment prepared from the H. lus amylollquefacjens) strain,
A new plasmid pBamHI RM23 is obtained.

The preparation process and restriction enzyme map of the pBamHI plasmid are shown in FIG. As is clear from Figure 1, this plasmid contains HIn at the restriction enzyme site of pTB53 plasmid.
Bacillus amyloliquefacins (B
acillus amyloliquefaciens
) It is a circular molecule with 22 Kb base pairs into which a DNA fragment containing the BamHI restriction endonuclease gene of strain H is incorporated.

(b) Preparation of microorganisms The conjugate of the chromosomal DNA fragment and vector DNA thus obtained is subjected to known transformation methods, such as the Journal of Bacteriology.
of Bacteriology) 8141 (19
When introduced into the recipient microbial cell by the competent cell method described in 81), desired genetic traits and vector D
A transformed strain having both NA traits can be obtained.

The recipient bacteria include the aforementioned Bacillus subtilis (Bacillus subtilis).
illus 5ubtth Is) MT2. M 111
Microorganisms commonly used in this type of technical field, such as No. 3, are advantageously used. A typical example is Bacillus subtilis strain M72 [Journal of Bacteriology]
l.

gy 831154 (1983)' genetic trait: tr
pC2, 1euc7, rmmm, Npr
) l.

There are currently two methods for selectively isolating clones of restriction system genes. The first method is to use bacteriophage infection (Proceeding of NationalAc
ademy 5science 781503. (198
1)) 6 In other words, the presence of a restriction/modification system in bacteria allows the system to resist infection by bacteriophages, so clones containing the cloned restriction/modification system genes should be protected against infection by phage. Selectively isolated as survivors from the library. The second method is to select clones of restriction genes by selecting modification genes (J
Our own of Bacterol.

gy 111i4501. (1985)). That is, by selecting a plasmid showing resistance to restriction endonucleases, a clone in which a modification system gene has been cloned is first selected. Next, since modification and restriction genes are often located close to each other, by cloning a larger region adjacent to the modification gene, a clone containing the restriction gene is selected. Any method can be used to select the B amH ■ restriction enzyme endonuclease clone according to the present invention.

(C) Restriction endonuclease production step (b) In order to culture the transformed strain obtained in step (b), a medium suitable for the production of the substance produced by specific genetic information and suitable for the growth of the host microorganism is used. However, in the method of the present invention, Bacillus subtilis (B.
An L-Broth medium (tryptone, yeast extract, salt) used as a growth medium for A. acillus 5ubcjlus may be used as a basic medium.

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

The culture method can adopt conditions such as I) H, temperature, and oxygen supply that are suitable for the growth of normal microorganisms of the genus Bacillus (order Bacillus). It is preferable to grow until the bacterial mass reaches the maximum, that is, until the late stage of logarithmic growth. The culture temperature is usually 30-37°C, and the pH condition is pH 5-37°C.
A range of 8, particularly around neutrality, is appropriate.

After collecting the obtained bacterial cells, they are extracted by centrifugation, ultrasonic disruption, etc., and then by combining nucleic acid removal method, salting out method, gel filtration method, ion exchange chromatography method, affinity chromatography method, etc. BamHI restriction endonuclease can be obtained.

(Effect of the invention) The plasmid of the present invention and the transformed strain introduced with the same are B.
In order to produce amHI restriction endonuclease in large quantities, it became possible to produce a large amount of 13 g mHI restriction endonuclease in the purification process.

EXAMPLES The present invention will be explained in more detail below with reference to Examples, but the present invention is not limited thereto.

(Example) (1) Preparation of vector plasmid pTB53 Bacillus carrying the DNA of vector plasmid pTB53
Subtilis (Bac order 1us 5ubtl order s) MT
-2 was prepared as follows and the vector plasmid pTB5
I got 3.

LB medium [Bacto-tryptone (DIf) per portion of pure water
co) lOg, Bacto Yeast Extract (Djfco
) 5g.

110g of NaC adjusted to pH 7.0) 100
Dispense ml into a 5001 volume Sakaro flask and incubate at 120℃ for 2 hours.
Autoclave sterilized for 0 minutes. Kanamycin 0.0004% and tetracytalin 0.0025% were added to this medium, and Bacillus subtilis carrying pTB53 DNA as a plasmid was grown.
MT-2 was inoculated into one platinum loop, cultured with shaking at 30°C for 16 minutes, and then centrifuged at 800 rpm for 5 minutes at 4°C to collect bacterial cells. Next, add 5+ag to the collected bacteria.
/+nl lysozyme (manufactured by Taiyo Kagaku ■), 50IIIM
Glucose, suspended in 25mM Tris-HCl buffer (pH 8,0) 51n+ containing 10mM EDTA, and incubated at 37°C.
It stood still for 30 minutes. Next, after leaving it in water for 5 minutes,
10 ml of 0.2N NaOH solution containing %SO5 was added, and the mixture was left standing in water for 10 minutes. Further, 7.5 ml of 3M sodium acetate solution (pH 4,8) was added, and the mixture was left standing in water for 15 minutes. Centrifugation was performed at 111i, 000 rpm for 120 minutes at 4°C.

Add 12 ml of Improper Tool to this supernatant, 10.
Centrifugation was performed at 18°C for 30 minutes at 000 rpm, and the resulting precipitate was washed with 70% ethanol.
It was dissolved in 6 ml of 50mM Lis-HCl buffer (pH 8.0) containing 50mM EDTA. Next, 5.9 g of cesium chloride and 0.3 ml of ethidium bromide solution (1% ethidium bromide solution containing 5% dimethyl sulfoxide) were mixed and centrifuged at 38,000 rpm for 140 hours at 18°C. The plasmid DNA layer was extracted with a syringe, ethidium bromide was removed by n-butanol extraction, and about 30 μg of pTB53 DNA was obtained by dialysis against 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA.

■ Preparation of chromosomal DNA carrying the BamHI restriction endonuclease gene
amyloles quefaclens) H strain to L-Bro
th medium [tryptone (formerly fcO) 10 per 11 pure water
5 g of g1 yeast extract and 110 g of NaC were inoculated into Co. 501, which had been adjusted to pH 7.0, and cultured with shaking at 30°C. Bacterial cells were collected 14 hours later. Next, the collected bacterial bodies are 1
0 mg/ml lysozyme [Taiyo Chemical Co., Ltd., suspended in 20 ml of 50IIIM Tris-HCl buffer (pH 7, 8) containing 20% sucrose and 1 mM EDTA, and allowed to stand at 37°C for 10 minutes. Next, 44ml of 0.1M EDTA solution (pH 9, B) containing 1% lauroyl sarcosinate
2.0 ml of a 5.44 mg/ml pronase solution was added thereto, and the mixture was allowed to stand at 50° C. for 30 minutes. Next, cesium chloride G
Ggs 10 mg/l ethidium bromide solution3.
31 and after mixing 38.000rpI1114
Centrifugation was performed for 0 hours. The DNA layer was extracted with a syringe, ethidium bromide was removed by n-butanol extraction, and the DNA layer was extracted with approximately
00 μg of chromosomal DNA was obtained.

(2) Insertion of Chromosomal DNA Fragment into Vector 1.0 unit of H1ndm restriction endonuclease was added to 3 μg of the chromosomal DNA obtained in (1), and the mixture was partially degraded by reaction at ℃ for 1 hour. Next, 1 μg of vector plasmid pT85 obtained in (1)
This was completely degraded by adding 4 units of )llnd■ restriction endonuclease and carrying out the reaction at 37°C for 11 hours.

3 μg of the chromosomal DNA fragment obtained by the above method and 1 μg of the pBR322 DNA fragment were mixed, and further Immuno.
Plasmid DNA incorporating chromosomal DNA was obtained by carrying out a ligation reaction at 15°C for 16 hours using 5 units of T4 phage-derived DNA callase in the presence of MATP and 5mM dithiothreitol.

(4) Transformation with a recombinant plasmid containing the BamHI restriction endonuclease gene Bacillus subtilis strain MT2 was inoculated into the L-Broth medium 51 and cultured with shaking at 37°C overnight. Next, add 1ml of it to T.
FI medium ((NH4)2SO40.2g per 11g of pure water
, K2HPO41,4g, KH2PO40, Eig
l Sodium citrate 0.1g1 Casamino acid 0.2
g, glucose 5 g1 MgSO40.2 g, arginine 0.05 g1) Wattfan 0.05 g) 201 and cultured with shaking at 37°C for 3 hours and 45 minutes. Furthermore, part 41
TFn medium ((NH4)2SO40 per pure water 11
,2g, K2HPO41,4gs KH2PO40,
6gs Sodium citrate 0.1g 1 force f / acid 0.1g, liquefaction: I-su 5g, MgSO40,
2g1 Arginine 0.005g, ) Watt Fan 0
．． 005g) was inoculated into Hml to prepare competent cells.

The plasmid DNA solution obtained in step (3) was added to 1 ml of the obtained competent cells, shaken vigorously at 37°C for 30 minutes, and then centrifuged (3,000 rpm 1
After collecting bacteria, 1 ml of the L-Broth medium was added and cultured with shaking at 37°C for 2.5 hours, and then spread on L-broth agar medium containing tetracycline and incubated at 37°C.
The cells were cultured overnight. The obtained transformed strain was transformed into a bacteriophage of Bacillus subtilis (order S) called ρ11C3 Fuji (Journa).
l of General Applied MI
crobology 25223. (1979)) was replicated in the LB medium containing 103-10' pfu. By selecting strains that grow on the same medium, we have established kanamycin and tetracycline resistance, and Ba.
Bacillus strains that produce mHI restriction endonuclease
Bacillus subtjljs
M T 2 (pB amHIRM22) (Feikoken Bibori No. 1θ175) was isolated.

■ Bacillus subtilis
Item IIs) B by MT2 (pBamHIRM23)
Production of amHI restriction endonuclease (transformant strain Bacillus subtilis MT2 obtained with small
(pBamHIRM23)
No.) was cultured with shaking at 37° C. for 16 hours in a 2-volume flask containing the L-Broth medium 5001. This was centrifuged to collect bacteria, and after washing, 10mM MgCl, 7
Suspended in 25 ml of 20 mM Tris-HCl buffer (pH 7,5) containing mM 2-mercaptoethanol,
Ultrasonic disruption was performed at 0°C for 10 minutes. Another 12.0
An enzyme extract was obtained by centrifugation at 0 rpm for 10 minutes. Next, ammonium sulfate powder was dissolved in this enzyme extract without addition of water cooling, and a 30 to 80% saturated fraction (saturation degree is expressed by the 0 sborne method) was collected by centrifugation. This recovered precipitate was dissolved in 2 ml of 10 mM phosphate buffer (pH 7,5) containing 2 mM mercaptoethanol and 5% glycerol, placed in a dialysis tube, and incubated overnight against 100 times the volume of the same buffer. Dialyzed. Next, a phosphocellulose (Whatman) column (capacity 2) equilibrated with the same buffer was added.
01). 0-1 after washing with 5 times the volume of the same buffer
．． OMKcl gradient elution was performed. BamHI
The restriction endonuclease was eluted at a KCI concentration of around 0.5M. Put the eluted enzyme solution into a dialysis tube and
A 0.21 enzyme solution was obtained by dialysis against a phosphate buffer containing M mercaptoethanol and 50% glycerol. When the enzyme activity of the obtained enzyme solution was measured, it was found to be to and ooo units, which is different from the parent strain Bacillus.
Bacillus amyl
The production amount was about 5 times that of the H strain.

The enzyme solution thus obtained did not contain other restriction endonucleases, phosphatases, nonspecific DNase, etc., and could be used in the field of genetic engineering. To measure the activity of Naori amHI restriction endonuclease, 1 μg of λ-DNA was mixed with 10 mM) Lis-HCl buffer, IOa+M magnesium chloride solution, 50 mM ammonium sulfate, and 7 mM 2-M pBamHIR.
Preparation process of M22 and restriction enzyme map R 1゜3゜

Claims (3)

[Claims] (1) Bacillus amyloliquefaciens
ns) Bacillus (
A novel recombinant plasmid that can replicate in bacteria of the genus Bacillus. (2) Bacillus amyloliquefaciens
ns) A novel microorganism belonging to the genus Bacillus transformed with a recombinant plasmid incorporating a chromosomal DNA fragment containing the BamHI restriction endonuclease gene derived from strain H. (3) Bacillus amyloliquefaciens
ns) Cultivating a microorganism belonging to the genus Bacillus that has been transformed with a recombinant plasmid incorporating a chromosomal DNA fragment containing the BamHI restriction endonuclease gene derived from strain H, and collecting BamHI restriction endonuclease from the culture. A method for producing a novel BamHI restriction endonuclease characterized by