SU1634713A1 - Method for preparing restriction endonuclease - Google Patents

Abstract

This invention relates to biochemistry and biotechnology, concerns a method for producing the restriction enzyme XBa I from the biomass of cells Xanthomonas badrii, strain VKPM B-2754 (ATCC 11672), and can be used in genetic engineering and molecular biology. The aim of the invention is to simplify the process while increasing the yield and quality of the target product. To do this, Xanthomonas badrii biomass is disrupted by ultrasound, the resulting cell homogenate is fractionated in a two-phase system containing 7% polyethylene glycol and 2% dextran in the presence of 0.35-0.42 M NaCI, the chromatographic purification of the preparation is carried out on heparin sepharose by applying the enzyme to an affinity sorbent equilibrated with YumM tris-HCl buffer with a pH of 7.2, followed by concentration of the target product by dialysis against a 50% glycerin solution. The yield of the restriction endonuclease XBa 1 is 12-15 thousand units / g biomass. The drug is free from impurities of nonspecific endo and enzonucleases (L

Description

This invention relates to biochemistry and biotechnology, concerns a method for producing the restriction enzyme XBa I from Xanthomonas badrii biomass, and can be used in genetic engineering and molecular biology.

The purpose of the invention is to simplify the process while increasing the yield and quality of the target product.

The method is carried out as follows.

The biomass of X.badrii cells, the strain VKPM B-2754 (ATCC 11672), is destroyed by ultrasound, then a concentrated mixture of polyethylene glycol and dextran in potassium phosphate buffer, pH 7.4, is added to the cell homogenate to a final concentration of 7% in polyethylene glycol mixture , dextran 2% and NaCI to concentration

0.35-0.42 M. After thorough mixing, the mixture is centrifuged at lOOOOg for 20 minutes, the upper polyethylene glycol phase containing the desired product is collected, diluted with buffer, heparin-sepharose, previously equilibrated with 10 mM Tris-HCl, pH 7.2, is poured. and this suspension is packed into the column (deposition in volume), and the proteins are eluted with a linear NaCI concentration gradient from 0 to 0.6 M in the buffer. The fractions containing endo-nuclease XA I are collected and concentrated by dialysis against a 50% glycerol buffer solution.

The output of the enzyme from 10 g of cells is 120-150 thousand units of act. The preparation is stored at -20 (± 2) ° C for 6 months without a decrease in activity. Free from significant impurities of nonspecific endonucleases: curing

with

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Treatment of phage T7 DNA with a 50-fold excess enzyme for 20 hours (1000-fold excess) at 37 ° C does not change the specific hydrolysis pattern.

The proposed method is illustrated by the following examples.

Example 1. All enzyme purification operations were carried out at 4 ± 4 ° C.

The activity of restriction enzyme XBa I is tested by the method of hydrolysis of DNA of phage T7, followed by separation of the obtained fragments by electrophoresis in a 0.8% agarose gel. The minimum amount of enzyme required for constant specific hydrolysis of 1 μg of DNA phage T7 for 1 h 37 ± 1 ° C was taken per unit of activity.

10 g of frozen biomass X. badrii VKPM B-2754 are suspended in 20 ml of 10 mM Tris-HCl, pH 7.9, containing 1 mM EDTA and 0.01 M 2-mercaptoethanol, triton X-100 is added to a final concentration of 0, 1% and sonicated at a frequency of 20 kHz and an amplitude of 17 ± 2 µm several times until the viscosity disappears in the biomass, periodically cooling the mixture to prevent the mixture from heating and inactivating the enzyme. Under continuous stirring, 30 ml of a mixture containing 21% polyethylene glycol and 6% dextran, 7.88 ml of 4M NaCI solution (to the final concentration of the latter: polyethylene glycol 7%, dextran 2%, NaCI 0.35 M) are poured to the cellular homogenate and 22.12 ml deionized water.

The mixture is continued to stir for 20 minutes, then centrifuged at 10,000 rpm for 20 minutes. The upper phase is taken, diluted v / fold with 10 mM Tris-HCl buffer, pH 7.2, containing 10 mM 2-mercaptoethanol, 0.1 mM EDTA and 10% glycerol (buffer A) and 10 ml of heparin is added. Sepharose, pre-equilibrated with buffer A. The mixture is stirred on a magnetic stirrer for 20 minutes and packed into a column (2.0 x 15 cm). The column is washed with 100 ml of 10 mM Tris-HCl, pH 7.5, containing 10 mM 2-mercaptoethanol, 0.1 mM EDTA and 10% glycerol (buffer B), the enzyme is eluted with a linear gradient of NaCl concentration from 0 to 0.6 M buffer B. Total gradient volume 100 ml. The rate of application, washing and elution is 15 ml / h. Collect fractions with a volume of 3 ml and analyze the activity of XAa I retrictase. Fractions containing the desired product, which elute in the range of NaCI concentration of 0.3-0.4 M, are pooled (v 15 ml), transferred to the dialysis bag and dialy - against 200 ml of 50% glycerol in 10 mM Tris-HCl, pH 7.4, containing 0.1 mM

EDTA 0.5 M KCI, 0.01 M 2-mercaptoethanol (buffer C), dialysis time 14-17 h.

The enzyme yield is 120 thousand units. Act. The enzyme concentration is 24,000 units. act./ml.

The resulting preparation of the restriction enzyme XBa I is stored at -20 ° C for 6 months without a decrease in activity. The content of impurities of nonspecific nucleases in the preparation was evaluated in two ways: by treating the DNA of phage 17 with excess enzyme for a long time and using the restriction-doping-re-restriction method. It was established that when processing 1 μg of DNA of phage T7 50 units of act. For 20 hours at 37 ° C, the XBa I restrictase drug does not change a clear specific pattern of hydrolysis. Fragments obtained by processing 1 μg of DNA phage T7 30 units. the act of preparation HBA I for 17 h at 37 ° C,

are ligated with DNA ligase of phage T4 not less than

than 90% and fully specifically hydrolyzed when treated with preparation XBa I.

Example 2 Restriction Endonuclease

XBa I is isolated from 10 g of cells in a similar manner.

Example 1, except that 9.45 ml of 4 M NaCI (to a final concentration of 0.42 M) and 20.55 ml of deionized water are added to the cell homogenate.

Impurities of non-specific endonucleases

and phosphatases are not detected in the preparation.

Claims (1)

The invention The method of obtaining the restriction enzyme XBa I, involving the destruction of the cells of the producer Xanthomonas badrli with ultrasound, the fractionation of the cell homogenate in a two-phase system containing 7% polyethylene glycol and 2% dextran, in the presence of NaCl, chromatographic purification of the sorbent, with a buffer. a solution of glycerol, characterized in that, in order to simplify the process while simultaneously increasing yield and quality of the target product, Xanthomonas badrli VKPM-2754 strain was used as a producer, cell homogenate was fractionated in the presence of 0.35-0.42 M NaCI, chromatographic purification of the enzyme was performed on heparinsepharose, balanced with 10 mM Tris-HCl buffer pH 7.2.