JPH037584A - Production of acci restriction endonuclease - Google Patents

Abstract

PURPOSE:To easily obtain the title endonuclease by transforming a host with recombinant plasmid incorporated with chromosome DNA fragment containing AccI restriction endonuclease gene of specific microorganism origin followed by culturing and then collecting the enzyme produced. CONSTITUTION:Chromosome DNA fragment containing AccI restriction endonuclease gene of Acinetobacter calcoaceticus origin is incorporated into plasmid, and using the resultant recombinant plasmid, host (e.g. Escherichia coli) is transformed and the resultant host is cultured to produce AccI restriction endonuclease. Thence, the objective AccI restriction endonuclease is collected from the cultured product. Thereby, mass production of the objective AccI restriction endonuclease can be easily accomplished without the need for removing both AccII and AccIII restriction endonucleases because of effecting no production thereof.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a new recombinant plasmid incorporating a chromosomal DNA fragment containing the Ace I restriction endonuclease gene, a host transformed by introducing the plasmid, and a host transformed with the plasmid. The present invention relates to a method for producing Ace I restriction endonuclease by culturing a host.

(Prior art) Type 1 restriction enzymes are highly specific enzymes that recognize and cleave a specific base sequence in deoxyribonucleic acid (DNA) tN. It is widely used in various fields.

To date, approximately 100 types of type restriction enzymes have been discovered and commercialized from small surfaces. Ace of the present invention
1m-limited endonuclease is also one of these ■-type restriction enzymes, and is an enzyme that recognizes and cleaves Φ GT AGCTCT in the base sequence of DNA.
obacfer cafecoaceticus) (Nucleic A
c1ds Re5earch L1r16s (1985
)).

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

That is, ■ Contains no other restriction enzymes.

■Does not contain phosphatase.

■Contains no non-specific DNase.

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

Therefore, commercially available restriction enzymes are
The Acc of the present invention is purified to a high purity by a combination of salting-out method, gel filtration method, ion exchange chromatography method, affinity chromatography method, etc.
The same method as for other restriction enzymes has been attempted for Ace I restriction endonuclease, but especially for Ace I restriction endonuclease, Ace
aceticus) uses CG CG in addition to Ace I restriction endonuclease. alIli, the restriction enzyme that cuts this, Qcc I! It was extremely difficult to eliminate this enzyme because it simultaneously produced Accm, a restriction enzyme that recognizes and cleaves TCCGGA and TCCGGA.

(Problems to be Solved by the Invention) The present inventors solved the drawback of the above method in that ^ccI [restriction endonuclease] and ^ccT[(restriction endonuclease) are simultaneously produced, and Create a strain that produces only
I conducted extensive research. As a result, the Acinetobacter
Acinetobacter ca
lcoaceticus) to the chromosome tIN containing the Acc I restriction endonuclease gene, inserted this into a vector to create a recombinant plasmid, and succeeded in obtaining a transformed host by introducing the plasmid. The host is 11cc! The present invention was completed based on this new knowledge.

(Means for Solving the Problems) That is, the present invention provides Acinetobacter calcoaceticus (A
cinetobacter calcoacetiqu
a recombinant plasmid incorporating a chromosomal DNA fragment containing the Acc I restriction endonuclease gene derived from s);
A method for producing Hecal restriction endonuclease, which comprises a host transformed with the plasmid, and culturing the host, and collecting Acc I restriction endonuclease from the culture.

Since the recombinant plasmid of the present invention and the host into which it is introduced produce only Acc I restriction endonuclease, there is no need to remove Acc I restriction endonuclease and Ace m restriction endonuclease in the purification process, and a large amount of Acc 1 restriction endonuclease can now be easily produced.

The present invention will be explained in detail below.

(a) Plasmids and their preparation The recombinant plasmids of the present invention contain forms that can proliferate in cultured cells, such as the colicin El factor, which is known as an extrachromosomal gene (plasmid) of microorganisms belonging to the genus Escherichia. Acinetobacter is added to the plasmid to be taken.
Acinetobacter ca
This is a plasmid that incorporates the [lN^ fragment] containing the Acc I restriction endonuclease gene derived from P. lcoaceticus.

The vector DNA may be one extracted from naturally occurring DNA or one in which part of the DNA other than the part essential for proliferation is missing, such as Co1B strain, pMB9 strain, pBR322 strain, etc. strain, pscl
Examples include the ol strain, the R6 strain, and the lambda phage strain.

In addition, any known method can be used to incorporate the chromosomal DNA fragment into the vector DNA. For example, chromosomal DNA is cut at a specific site by treatment with an appropriate restriction enzyme (Endonuclease), and then treated in the same manner. A method is used in which the vector DNA is mixed with the vector DNA and religated using ligase.

Using pUc19 plasmid as vector DNA,
This is supplemented by Acinetobacter calcoaceticus (Acinetobacter calcoaceticus).
A novel plasmid pAcc I RM 8 is obtained by incorporating a chromosomal DNA fragment prepared from Tobacter calcoacaticus).

The preparation process and restriction enzyme map of the pAcc I RM 8 plasmid are shown in FIG. 1. As is clear from FIG.
This plasmid contains Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) at the Bam+Hz site of the restriction enzyme site of the pUc19 plasmid
It is a circular molecule with 8 kb base pairs into which a DNA fragment containing the Ace I restriction endonuclease gene of S. ceticus has been incorporated.

Φ) Preparation of microorganisms The conjugate of the chromosomal DNA fragment and vector DNA obtained in this way is subjected to a transformation method, such as sit-transformation.
When introduced into a recipient microbial cell by the shot gun method, a transformed strain having both the desired genetic traits and the traits of the vector DNA can be obtained.

The recipient bacteria include the aforementioned Escherichia coli DB101.
, C600, DP, 5upF, x1776, LH392, etc., which are commonly used in this type of technical field, are advantageously used. A typical example is the Escherichia coIJFIBIOI strain [Molecular Cloning A Laboratory Manual,
(Molecular Cloning A Lab
oratory Manual P, 504 (19B2
) Reference; genetic trait F-2hsd s20 (r-1+1l
l-1) reCA13, ara, 14, pro A2
, facYl, gal K2, rps L20 (Sm'
), xyl-5, +*tj! -1, sup E44λ-
)].

The microorganism obtained by the transformation method by introducing the plasmid pAcc I RM8 into this Escherichia coli BIOI strain is a new microorganism, and Escherichia coli HBIOI (pAcc IRM8) (Escher
ichia Co11HBIOI (pAcc I RM
8)) and was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology on May 25, 1989, and its deposit number is FERM P-10740 (FERM P-1074).
The mycological properties of Escherichia coli HBIOI (pAcc T RM8) obtained in this way, which is
Comparing the properties of the I strains, they are completely identical except that the former has the ability to produce Acc I restriction endonuclease and ampicillin resistance, whereas the latter does not have these properties.

(C) To culture the transformed strain obtained in the restriction endonuclease production step (b), use a medium suitable for the production of the substance produced by specific genetic information and suitable for the growth of the host microorganism. However, in the method of the present invention, LB medium (tryptone, yeast extract, salt), which is usually used as a growth medium for Escherichia coli, can be used.
, BPB medium (Dirco; polypeptone, yeast extract, potassium phosphate), nutrients/agar, medium (Dirc
o 0001) A tryptone/salt medium or the like prepared as a basic medium may be used.

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

The culture method can use conditions such as pH, temperature, and oxygen supply that are suitable for the growth of normal Escherichia microorganisms. It is preferable to grow until the maximum amount is reached, that is, to the late stage of logarithmic growth, and the culture temperature is usually 3.
The suitable pH conditions are p) 15 to 8, particularly around neutrality.

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. Acc I restriction endonuclease can be obtained.

(Effect of the invention) The plasmid of the present invention and the transformed strain into which it is introduced are A
To produce only ce I restriction endonuclease,
In the purification process, there is no need to remove Acc II restriction endonuclease and Acc m restriction endonuclease, which makes it possible to easily produce a large amount of hecal restriction endonuclease.

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 (1) Preparation of chromosome [INA] carrying the gene for Accl restriction endonuclease Acinetobacter calcoaceticus (Acinet).

bacter calcoaceticus)
Roth medium [Pure 11 per tryptone (Dirco
) 10 g-, yeast extract 5 g, NaCl L
og adjusted to pH 7.0) inoculated into 50d,
Shaking culture was performed at 0°C. After 14 hours, the bacterial cells collected at the 0th stage were mixed with 10 μ/ml of lysozyme [manufactured by Taiyo Kagaku ■] and 20% Sea II! , 50mM containing 1nM EDTA) was suspended in 20d of Lis-HCl buffer (pH 7,6) and allowed to stand at 37°C for 10 minutes.
) 44d and 5.44■/IIi pronase solution 2.
0 m was added and left at 50°C for 30 minutes.

Next, 66 g of cesium chloride and 3.3 d of ethidium bromide solution of 10 μ/d were added, and after mixing, 38.00 O
Centrifugation was performed at rpm for 40 hours. The OIA layer was removed with a syringe, ethidium bromide was removed by n-butanol extraction, and 100% solution containing 1 mM EDTA was added.
Approximately 380 μg of chromosomal DNA was obtained by dialysis against 5M) Lis-HCl buffer (pH 8,0).

(2) Insertion of chromosomal DNA fragment into vector (1)
Add 0.02 units of 5au3^I restriction endonuclease to 3 μg of the chromosomal DNA obtained in
This was partially decomposed by carrying out a reaction at 7°C for 1 hour.

Next, vector plasmid pUc19 (manufactured by Toyobo ■)
For 1 μg, 4 units of BamHI restriction endonuclease were added and the reaction was carried out for 1 hour at 37°C to completely decompose it. Furthermore, 1 unit of alkaline phophatase was added and the reaction was carried out for 11 hours at 37°C. The 5' terminal phosphoric acid was removed.

Mix 3 μg of chromosome [INA fragment obtained by the above method and 1 Mg of pBR322 DNA fragment, and add 1
5 in the presence of mMATP and 5mM dithiothreitol.
Plasmid DNA incorporating chromosomal DNA was obtained by carrying out a ligation reaction at 15°C for 516 hours using DNAIJ gauze derived from UNIT's T4 phage.

(3) Transformation with a recombinant plasmid containing the Accl restriction endonuclease gene Escherichia coli strain 112 and Escherichia 398
Escherichia 117881, a hybrid strain of the strain
01 strain (manufactured by Invitrogen) in LB medium [pure water 12
10 g of tryptone (Dirco), 5 g of yeast extract, and 5 NaCI Log were inoculated into a 10-ml strain prepared to pH 1.0, cultured with shaking at 37°C, and grown to the logarithmic phase, followed by harvesting. This was suspended in a solution with a final concentration of 0.03M CaCl□ under water cooling to obtain competent cells. Add the solution of plasmid 1) HA obtained in (2) to this cell suspension, react for 60 minutes under water cooling, and apply heat syrup at 42°C for 1 to 2 minutes to transfer the plasmid DN^ into the cells. Then, this cell suspension was separately inoculated into the LB medium, and 37
After performing a transformation reaction by culturing with shaking for 13 to 5 hours at °C, a strain having ampicillin resistance and producing ccl restriction endonuclease was isolated, and Escherichia coli HBIOI (pAcc [RM8) 1Q'740).

(4) Escherichia co'J HBOI (pAc
Production of Acc 1 restriction endonuclease using c I RM8) (3)
Coli HBOI (pAcc I RM8) (Feikoken Bibori No. 10740) was added to the LB medium containing 500 d of the above.
Culture was carried out with shaking at 37°C for 116 hours in a 1-volume flask. The bacteria were collected by centrifugation, and after washing, 1O+*M M
gC1□, 71IIM2-20mM Tris-HCl buffer (pH 7,5) containing mercaptoethanol 25m
The suspension was suspended in water and subjected to ultrasonic disruption at 0°C for 10 minutes.

Further, an enzyme extract was obtained by centrifugation at 12,000 rpm for 10 minutes.

Next, ammonium sulfate powder was added and dissolved in this enzyme extract under water cooling.
A 0 to 80% saturated fraction (the degree of saturation is expressed by the 0sborne method) was collected by centrifugation. 2 m of this collected sediment
M mercaptoethanol, 1 containing 5% glycerol
0mMIJ acid buffer (pH 7,5) '2 rtdl
The solution was dissolved in a dialysis tube, and the solution was dialyzed against 100 times the volume of the same buffer. Next, a column of phosphocellulose (Whatman) equilibrated with the same buffer (
Capacity 20-) was adsorbed. After washing with 5 times the volume of the same buffer, 0-1.0M MCI gradient elution was performed. B
an1[I restriction endonuclease was used at an MCI concentration of 0.5
It was eluted around M. The eluted enzyme solution was put into a dialysis tube and dialyzed against a phosphate buffer containing 2mM mercaptoethanol and 50% glycerol to obtain an enzyme solution of the 5th relative. The enzyme activity of the obtained enzyme solution was measured and found to be 500 units.

The enzyme solution thus obtained did not contain other restriction endonucleases, phosphatases, non-specific DNase, etc., and could be used in the field of genetic engineering.

The activity of Acel restriction endonuclease was measured by dissolving 1 μg of λ-DNA in 45 μ2 of a reaction solution consisting of 20++M Tris-HCl buffer, 10+M magnesium chloride solution, 50mM ammonium sulfate, and 7mM 2-mercaptoethanol, and mixing the mixture. After adding 5μ2 of the enzyme solution to the solution and performing a reaction at 37°C for 1 hour, the measurement is performed by performing agarose electrophoresis.

One unit of enzyme activity refers to the enzyme activity that completely degrades 1 μg of λ-DNA in 1 hour at 37° (pH 7.5).

[Brief explanation of drawings]

Figure 1 shows the preparation steps and restriction enzyme map of pAcc I RM8.

Claims (3)

[Claims] (1) Acinetobacter calcoaceticus (Acine
A recombinant plasmid incorporating a chromosomal DNA fragment containing the AccI restriction endonuclease gene derived from Tobacter calcoaceticus. (2) Acinetobacter calcoaceticus (Acine
A host transformed with a recombinant plasmid incorporating a chromosomal DNA fragment containing the AccI restriction endonuclease gene derived from Tobacter calcoaceticus. (3) Acinetobacter calcoaceticus (Acine
AccI restriction method comprising culturing a host transformed with a recombinant plasmid incorporating a chromosomal DNA fragment containing the AccI restriction endonuclease gene derived from Tobacter calcoaceticus) and collecting AccI restriction endonuclease from the culture. Method for producing endonuclease.