RU2136749C1 - Method of restriction endonuclease producing - Google Patents

Abstract

FIELD: biotechnology, biochemistry, microbiology. SUBSTANCE: method involves cells disruption by ultrasonic oscillation, thermal treatment at 58-60 C for 5-10 min, not less, centrifugation, chromatography on DEAE-cellulose (DE-52), an additional purification on phosphocellulose P-11, heparin-Sepharose and the concentration against 50% glycerol solution. The yield of enzyme is 170 000-200 000 U/10 g of wet biomass. Method ensures to obtain the enzyme no containing impurities of nonspecific nucleases and phosphatases and at the low level of protein. EFFECT: improved method of producing. 4 cl, 1 tbl, 2 dwg, 4 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 Haemophilus, in particular to the production of Hinf I restriction endonucleases that recognize and cleave the 5'-GANTC-3 'nucleotide sequence.

 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 well-known methods of purification of restriction endonucleases are the methods listed at the end of the description in the table.

 A known method for the isolation of restriction endonuclease Hinf I [4], which includes a small number of stages of isolation, however, it is designed to obtain small samples of the enzyme. It is not technologically advanced and cannot be used when scaling the process in the form of the difficulty of controlling the temperature in the process of destruction and heat treatment of biomass, which requires additional material equipment.

 A known method of isolating restriction endonuclease from a significantly larger amount of Haemophilus influenzae cell biomass [3, prototype], which includes the following stages: destruction of cells by ultrasound (obtaining a coarse extract), centrifugation and release from debris, purification on hydroxyapatite, ammonium sulfate precipitation, dialysis, purification on Sephadex G-25; chromatography on P-11 phosphocellulose; concentration on lipogel.

 The disadvantage of this method is the multi-stage process. In addition, the method leads to large losses and makes it possible to obtain about 8% of the total activity of the enzyme, the coarse extract contains a large number of non-specific nucleases.

 An object of the invention is to improve the process of isolation of the restriction endonuclease Hinf I to increase the yield of high purity product.

 The problem is solved by changing the heat treatment conditions and improving the chromatographic purification of the product on DEAE-cellulose-DE-52.

SUMMARY OF THE INVENTION The biomass of the cells of the strain H. influenzae stored in the collection of the Research Institute of Microorganism Culture Collection "Vector" is destroyed under stationary conditions using ultrasound, then the suspension is kept at a temperature of 58 - 60 ^o C for 10 min, cooled and freed from cell debris by centrifugation. Purification on DEAE-cellulose-DE-52 is carried out by mixing the enzyme fraction with the sorbent in buffer A containing 0.2 M NaCl (1: 1 ratio).

The bacteria cells of H. influenzae (the required amount) are suspended in buffer A (0.01 M potassium phosphate buffer pH 7.5, 1 mM 2-mercaptoethanol, 0.1 mM EDTA) at a rate of 2 ml per 1 g of biomass and ultrasonically disintegrated at a temperature of 4 ^o C. After which the suspension is kept at a temperature of 58 - 60 ^o C for 10 minutes, which allows to reduce the protein content by at least 25 - 30%, then the extract is freed from debris by centrifugation, and the supernatant is used for further purification on DEAE cellulose -DE-52. Fractionation on DEAE-cellulose-DE-52 is carried out in buffer A in the presence of NaCl to a final concentration of 0.2 M. DEAE-cellulose-DE-52 is filtered on a glass filter under the discharge of a water-jet pump, and P11 phosphocellulose is applied to the enzyme. Elution of sorbed proteins is carried out by a linear gradient of NaCl concentration from 0.2 to 0.6 M in buffer A. The obtained fractions are examined for the presence of restrictase, combined and used for the next purification step on heparin-sepharose. The elution of sorbed proteins after dialysis is carried out by a linear gradient of NaCl concentration 0-0.6 M in the buffer. Fractions giving complete specific hydrolysis are combined and concentrated by dialysis against a 50% glycerol solution in buffer A. The enzyme yield from 10 g of cells is 170,000 - 200,000 units. Act. , which is 30 - 35% of the total activity of the enzyme. The drug is stored at minus 20 (± 2) ^o C for at least 12 months without a decrease in activity. The enzyme is free from significant impurities of non-specific endonucleases: treatment of lambda phage DNA with a 10-fold excess of the enzyme for 16 hours (160-fold excess) at 37 ^° C does not change the specific pattern of hydrolysis (Fig. 1).

 This method allows to increase the yield of the enzyme preparation by an average of 4 times.

Significant differences of the invention are the following:
- conducting heat treatment at a temperature of 58 - 60 ^o C for at least 5 to 10 minutes after processing the cell suspension with ultrasound;
- chromatographic purification on DEAE-cellulose-DE-52.

 From literary sources it is known that heat treatment of cell suspension leads to better cleaning of cell debris [4]. However, as shown in the experiment, the simultaneous heating and destruction of the cell suspension by ultrasound does not allow for proper control of the heating temperature, which leads either to inactivation of the enzyme when the temperature rises or to a deterioration in purification at a low temperature.

 The combination of heat treatment with chromatography on DEAE-cellulose-DE-52 leads to significant purification of the cell suspension, which reduces the number of stages of further processing.

 Since the proposed method for producing restriction endonuclease Hinf I was proposed for the first time, we can conclude that the proposed strain meets the criteria of the invention of "novelty" and "inventive step".

In the figure 1:
1 lane - hydrolysis of 2 μg lambda phage DNA with a 10-fold excess of the enzyme for 1 hour;
Lane 2 — hydrolysis of 2 μg lambda phage DNA with a 10-fold excess of the enzyme for 16 hours;
Lane 3 - DNA control of phage lambda.

In figure 2:
1 lane - hydrolysis of 2 μg lambda phage DNA for 1 hour with a 10-fold excess of the enzyme;
2 lane - 2 μg of DNA fragments crosslinked by DNA ligase;
Lane 3 — hydrolysis of cross-linked DNA fragments with restriction enzyme Hinf I;
Lane 4 - DNA control of phage lambda.

 The invention is illustrated by the following examples of specific performance.

Example 1. Isolation of Hinf I restriction endonuclease I. 10 g of the biomass of H.influenzae cells are suspended in 20 ml of 0.01 M Tris-HCl buffer, pH 7.5, containing 0.01 M 2-mercaptoethanol (the extraction buffer is voiced on a UZDI-2T 8 disintegrator every 30 seconds, then the cellular hematogen is heated at a temperature of 58 - 60 ^o C for 10 minutes, centrifuged for 40 minutes at 18000 rpm, the resulting supernatant is fractionated on DEAE-cellulose-DE-52 in 0.01 M potassium phosphate buffer, pH 7.5, containing 0.01 M 2-mercaptoethanol 1 MM EDTA (buffer A) in the presence of 0.2 M NaCl. DEAE-cellulose-DE-52 is filtered off glass filter under a water-jet pump discharge. The clarified extract is applied to a P-11 phosphocellulose column balanced with 0.2 M NaCl in buffer A. Sorbed proteins are eluted with a linear gradient of NaCl concentration from 0.2 to 0.6 M in buffer A at a rate of 20 ml / h, the volume of the gradient is 300 ml, The fractions giving a complete specific hydrolysis are combined.The enzyme is eluted at a concentration of sodium chloride of 0.3 M. To liberate the fraction from sodium chloride, dialysis in buffer A is carried out for 2 hours and applied to heparin-sepharose, previously equilibrated with buffer A. The sorbed proteins are eluted with a linear gradient of NaCl concentration in buffer A from 0 to 0.6 M. The total volume of the gradient is 140 ml. Fractions containing the restriction endonuclease Hinf I, are combined and dialyzed against 50% glycerol in buffer A. The enzyme is stored at a temperature of minus 20 ^o C for at least 12 months without losing activity. The yield is 170,000 ea. The concentration of the enzyme is 60,000 ea / ml. The content of impurities of nonspecific nucleases in the preparation is evaluated in two ways: by processing the lambda phase DNA for a long time with an excess of the enzyme and by the restriction – ligation – repeated restriction method (Fig. 1, 2).

Example 2. Determination of enzyme activity. Hinf I restriction endonuclease activity was tested by hydrolysis of lambda phage DNA, followed by separation of the obtained fragments by electrophoresis on a 1.4% agarose gel. For the unit of activity of the enzyme take the minimum amount of enzyme necessary for complete specific hydrolysis of 1 μg of lambda phage DNA for 1 h at 37 ^o C.

 Example 3. Isolation of restriction endonuclease with an additional step on hydroxylapatite. Isolation is carried out according to the same scheme as in example 1, but with additional post-treatment with hydroxylapatite. The yield of the enzyme fell by 50%, which is probably due to the very low protein content in the preparation of the enzyme and its purity.

Example 4. Isolation of Hinf I restriction endonuclease. Isolation is carried out as in Example 1, but without heating the supernatant at 60 ^° C. The total enzyme activity is 64,000 units / 10 g of biomass. An additional purification step is required due to the poor quality of the enzyme (protein content is 4.8 mg / ml).

List of references
1. Lebedev L. R., Pustoshilova N. M. // Abstract. conf. Preparative scalable chromatography of biologically active substances and alternative methods. L., 1991.S. 89 - 92.

 2. Lebedev L.R., Andreeva I.S., Afinogenova G.N. et al. // Biological Sciences. 1992. N 2.P. 33 - 40.

 3. Kopecka H. // Bioch. et Bioph. Acta, 1975, v. 391, p. 109 - 120.

 4. Puchkova L. I., Serov G. D. A method of producing restriction enzymes. A.S. USSR N 1406159.

Claims (4)

 1. The method of obtaining restriction endonuclease Hinf I, including destruction of cells by ultrasound, centrifugation, chromatography and concentration, characterized in that the cell suspension is subjected to heat treatment, chromatography is performed on DEAE-cellulose-DE-52, purified on phosphocellulose R-11, heparin-sepharose , concentrate against a 50% glycerol solution. 2. The method according to claim 1, characterized in that the heat treatment is carried out for at least 5-10 minutes at 58-60 ^° C after the destruction of the cells by ultrasound. 3. The method according to claim 1, characterized in that the chromatography on DEAE-cellulose-DE-52 is carried out by mixing it with buffer A containing 0.2 M NaCl, keeping for 1 h at 0 - 4 ^o C.  4. The method according to claim 3, characterized in that the chromatography on DEAE-cellulose-DE-52 is carried out by mixing the enzyme fraction with the sorbent in buffer A containing 0.2 M NaCl in a 1: 1 ratio.

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