RU2233877C2 - Method for preparing endonuclease restriction sst 12i - Google Patents

Abstract

FIELD: biochemistry, microbiology, genetic engineering.

SUBSTANCE: invention relates to isolation and purification of site-specific endonuclease restriction synthesized by microorganisms of genus Streptomyces, namely, to preparing site-specific endonuclease restriction Sst 12I that recognizes and cleaves the nucleotide sequence: 5'-CTGCAG-3'. Method involves disruption of cells by ultrasonic oscillation, fractional thermal treatment (3 times for 3 min) at temperature 60 C, not above, centrifugation, chromatography on phosphocellulose P-11 column and concentrating against 50% glycerol solution. The yield of enzyme is 500000 U/10 g of biomass. This method provides preparing enzyme without impurities of nonspecific nucleases and phosphatases.

EFFECT: improved preparing method.

3 dwg, 3 ex

Description

The invention relates to biochemistry and microbiology, in particular to the isolation and purification of restriction endonucleases. synthesized by microorganisms of the genus Streptomyces, namely, the production of restriction endonucleases Sst 12I, which recognizes and cleaves the nucleotide sequence 5’CTGCAG3 ’.

Site-specific restriction endonucleases (restrictase) have become an indispensable tool for studying the structure of DNA, the creation of recombinant molecules, and the study of genetic material. In this regard, the urgent task is to create methodological approaches to the development of highly efficient technology for producing enzymes of this type.

In each case, this problem is solved empirically by selecting methods and conditions for the purification of enzymes during preliminary experiments. The most famous methods of purification of restriction enzymes are described in [1, 2].

A known method of producing restriction endonuclease Hinf I [1], which includes the destruction of cells by ultrasound, heat treatment for at least 5-10 minutes at 58-60 ° C, centrifugation, chromatography on DEAE-cellulose-DE-52. tertiary treatment on R-11 phosphocellulose, heparin-sepharose, concentration against a 50% glycerol solution. The yield of the enzyme is 170,000-200,000 units / 10 g of crude biomass.

The disadvantages of this method of obtaining are multi-stage process (the use of three sorbents, which leads to the loss of the drug), a decrease in activity with prolonged heating and lengthening the time of isolation of the enzyme.

A known method of producing restriction enzymes (3, prototype], which includes disintegration by ultrasound at a temperature increase of 50-60 ° C for 10-15 minutes, centrifugation, chromatography.

The disadvantage of this method of isolation of restriction enzymes is that the temperature control mode is not suitable for all restriction enzymes, i.e. long exposure at 50-60 ° C often leads to inactivation of enzymes secreted from mesophilic strains of microorganisms; at the subsequent stage of chromatographic purification, the enzyme is lost due to incomplete sorption of the protein on phosphocellulose.

An object of the invention is to improve the process of isolation of restriction endonuclease Sst 12I to increase the yield of the product with a high degree of purity and to extend its shelf life.

The problem is solved by using the method of producing restriction endonuclease Sst 12I, including the destruction of cells by ultrasound, heat treatment at a temperature not exceeding 60 ° C, centrifugation, chromatography on phosphocellulose R-11, concentration against a 50% glycerol solution. Heat treatment of the cell suspension is carried out fractionally for at least three stages of 3-5 minutes each and after each stage of the heat treatment, the suspension is cooled to 3-4 ° C. Centrifugation of the suspension will be performed after each heat treatment step at 15,000 rpm for 30 minutes. The process of obtaining the enzyme proceeds at a pH of at least 7.4-7.5, and before the chromatography step on P-11 phosphocellulose, the pH of the suspension is reduced to 7.0.

The enzyme is a viscous solution in a 50% glycerol solution containing 10 mm cadmium phosphate pH 7.0; 100 mM sodium chloride; 1 mM EDTA; 7 mM 2-mercaptoethanol.

The concentration of protein in solution is not less than 0.3 mg / ml, specific activity is not less than 50,000 U / mg of protein, activity is not less than 10,000 U / ml.

The exact molecular weight of the enzyme has not been established.

The enzyme requires Mg ^{2+ ions} at a concentration of 5 mM, 2-mercaptoethanol at a concentration of 7 mM, or dithiothreitol at a concentration of 1 mM.

Optimum pH of action 8.0

The optimum temperature is 55 ° C.

The optimum salt NaCl 50 mm.

A salt concentration of more than 100 mM inhibits the activity of restrictase.

The restriction enzyme Sst 121 hydrolyzes double-stranded DNA molecules in a strictly defined sequence: 5’-CTGCAG-3 ’. As a result of the action of the enzyme, double-stranded DNA fragments are formed that have 3'-hydroxyl and 5'-phosphate ends.

To isolate the restriction enzyme, the biomass of the cells is suspended in buffer A at the rate of 2 ml per 1 g of biomass and disintegrated at a temperature of 4-5 ° C. Then the suspension is subjected to heat treatment. The heat treatment is carried out fractionally: the suspension is maintained at a temperature of 55-60 ° C three times for 3-5 minutes with centrifugation after each temperature control. Under this heat treatment mode, stabilization of the protein molecule of the enzyme and liberation from most of the impurity proteins are observed. With continuous processing for 9-10 minutes, the secreted enzyme is inactivated.

The whole process of obtaining the product (except for chromatography) proceeds at pH 7.5, this is due to the most optimal condition for the destruction of cell walls and the release of the enzyme, a sharper increase in the pH of the medium leads to a shock state of the cells and inactivation of the protein.

Further purification of the enzyme is carried out by chromatography on phosphocellulose R-11. Before applying to the column, the pH of the enzyme extract is changed from 7.5 to 7.0. Under such conditions, the enzyme is completely sorbed on phosphocellulose. Sorbed proteins are eluted using a linear NaCl gradient from 0 to 0.6 M in buffer B. Fractions giving a complete specific hydrolysis are combined and concentrated by dialysis against a 50% glycerol solution in buffer B.

The enzyme yield from 10 g of cells is 500,000 E. The enzyme obtained in this way is free of nonspecific endonuclease impurities: treatment of phage DNA with a 10-fold excess of the enzyme for 16 hours at 37 ° C does not change the specific picture of hydrolysis (Fig. 1).

Checking the obtained preparation of the enzyme for purity shows that the resulting preparation does not also contain impurity exonuclease and phosphotase activities (Fig. 2) and is suitable for molecular biological and genetic engineering work.

The restriction enzyme Sst 12I is stored at -20 ° C for 1-10 years without loss of activity (figure 3).

New in comparison with the prototype is:

- the use of fractional three-fold heat treatment at a temperature not exceeding 60 ° C for 3 minutes, followed by centrifugation after each temperature control to stabilize the protein molecule and free most of the impurity proteins;

- lowering the pH from 7.5 to 7.0 before carrying out the chromatography step for complete sorption of the protein on R-11 phosphocellulose.

Since we offer no way to obtain restriction endonuclease Sst 12I proposed for the first time, we can conclude that the proposed strain meets the criteria of the invention of "novelty" and "inventive step".

The method is illustrated by the following graphic materials.

In the figure 1:

1 lane - hydrolysis of 1 μg DIK phage λ with a 10-fold excess of the enzyme for 1 h;

Lane 2 — hydrolysis of 1 μg phage DNA λ with a 10-fold excess of the enzyme for 16 hours;

Lane 3 - control of phage λ DNA.

In the figure 2;

1 lane - hydrolysis of 1 μg of phage λ DNA for 1 h with a 10-fold excess of the enzyme;

2 lane - 1 μg of DNA fragments crosslinked by DNA ligase;

Lane 3 — hydrolysis of cross-linked DNA fragments by restriction enzyme Sst 12I.

In figure 3:

1 lane - hydrolysis of 1 μg of phage λ DNA with a single excess of the enzyme, shelf life 0 years;

Lane 2 — hydrolysis of 1 μg of phage λ DNA with a single excess of the enzyme, shelf life of 5 years;

Lane 3 - hydrolysis of 1 μg of phage λ DNA with a single excess of the enzyme, shelf life of 8 years.

The following are examples of specific implementation of the method.

Example 1, Isolation of Sst 12I restriction enzyme. To isolate the restriction enzyme, the bacterial biomass of the strain Streptomyces sp. ST-12 in an amount of 10 g was suspended in buffer A (0.01 M potassium phosphate buffer pH 7.5, 1 mm 2-mercaptoethanol, 0.1 M EDTA) at a rate of 2 ml per 1 g of biomass and subjected to ultrasonic disintegration on a disintegrator (MSE, England) at a temperature of 4-5 ° C. Then the suspension is subjected to heat treatment. The heat treatment is carried out fractionally: the suspension is maintained at a temperature of 55 ° C three times for 3 min, with centrifugation after each temperature control at a temperature of 3-4 ° C. Centrifugation mode: speed 15000 rpm, 30 min on a J-21 centrifuge (Beckman, USA). Further purification of the restriction enzyme Sst-12I is carried out by chromatography on phosphocellulose R-11 (Whatman, England). Before applying to the column, the pH of the enzyme extract is changed from 7.5 to 7.0. The sorbed proteins are eluted with a linear NaCl gradient from 0 to 0.6 M in buffer B (0.01 M potassium phosphate buffer pH 7.0, 1 mM 2-mercaptoethanol, 0.1 M EDTA,). Fractions giving complete specific hydrolysis are combined and concentrated by dialysis against a 50% solution of glycerol in buffer B. The enzyme yield of 10 g of cells is 500,000 E. The enzyme obtained in this way is free from impurities of non-specific endonucleases: treatment of phage DNA with a 10-fold excess the enzyme for 16 hours at 37 ° C does not change the specific picture of hydrolysis (figure 1). Checking the obtained preparation of the enzyme for purity shows that the resulting preparation does not also contain impurity exonuclease and phosphotase activities (Fig. 2) and is suitable for molecular biological and genetic engineering work.

Example 2. Determination of the activity of restriction enzyme Sst 12I. The method for determining the activity of the restriction enzyme Sst 12I is based on determining the completeness of the specific hydrolysis of the native DNA molecules of phage C 1857 under optimal conditions for the enzyme: 100 mM Tris-Hcl buffer pH 8.0; 5 mM magnesium chloride; 50 mM NaCl; 7 mM 2-mercaptoethanol; temperature 55 ° C. To determine the activity of Sst 121, 20 μl of the reaction mixture containing 1 μg of phage C1857 is poured into numbered tubes and then 1 to 10 μl of the enzyme is added to each tube, kept for 1 h at 55 ° C. The hydrolysis products are separated electrophoretically in 1% agarose .

The unit of activity (E) is the minimum amount of the enzyme, which for 1 h under optimal conditions completely hydrolyzes 1 μg of phage λ C1857 in 20 μl of the reaction mixture.

Example 3. Determination of restriction enzyme activity Sst 12I after long-term storage. Samples of the Ssst 12I preparation after long-term storage are subjected to activity determination analysis. The restriction enzyme Sst 12I is stored at -20 ° C for 1-10 years without loss of activity. The method for determining the activity of long-stored batches of the enzyme is based on the determination of the complete specific hydrolysis of the native DNA phage λ C1857 molecules. Optimum conditions for hydrolysis of the substrate with restriction enzyme Sst 121: 100 mM Tris-HCl buffer pH 7.5; 5 mM magnesium chloride; 7 mM 2-mercaptoethanol; temperature 55 ° C. The hydrolysis reaction is carried out in 20 μl of the reaction mixture containing 1 μg of native DNA of phage C1857 at a temperature of 55 ° C (Fig.3).

The unit of activity (E) is the minimum amount of the enzyme, which for 1 h under optimal conditions completely hydrolyzes 1 μg of phage λ C1857 in 20 μl of the reaction mixture.

Thus, in comparison with the prototype, the product yield is increased 2.5 times, and its shelf life is at least 10 years.

Literature

1. RF patent No. 2136749 "Method for producing restriction endonuclease Hinf I", C 12 N 9/14, publ. BI 25, 1999

2. Bickle T.A., Pirotta V., Imber R. "A simple, general procedure for purifying restriction endonucleases" - In: Nucieic acids research, v. 4, No. 8, Aug. 1977.

3. USSR author's certificate No. 1406159 "Method for producing restrictase" C 12 N 9/16 publ. BI No. 24, 1988.

Claims (1)

A method of producing restriction endonuclease Sst 121, including ultrasonic disruption, heat treatment at a temperature not exceeding 60 ° C, centrifugation, chromatography on P-11 phosphocellulose, concentration against a 50% glycerol solution, characterized in that the cell suspension is heat treated fractionally, at least than in three stages of 3-5 minutes each, after each stage of the heat treatment, the suspension is cooled to 3-4 ° C and centrifuged at 15,000 rpm for 30 minutes, the entire process for producing the enzyme is carried out at a pH of at least 7.4-7, 5, and before the stage chrome atography on R-11 phosphocellulose the pH of the suspension is reduced to 7.0.

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