RU2070925C1 - Strain of bacterium escherichia coli - a producer of restriction endonuclease eco 71 ki - Google Patents

Abstract

FIELD: genetic engineering, biotechnology, biochemistry. SUBSTANCE: strain E. coli BM KB-200 6D is isolated from clinic isolates after screening strain-producers of restrictases. Enzyme activity is detected in extracts obtained from the separate colonies grown on agarized medium followed by electrophoresis of DNA cleavage fragments in agarose gel. Obtained strain is a producer of restrictase Eco 71 KI, isoschizomer or restriction endonuclease Eco 31 I. Enzyme content in the proposed strain is at least 200 000 U/g wet cell biomass, yield of the purified enzyme is 5000 U/g cell biomass. EFFECT: new strain indicated above as a restriction endonuclease producer.

Description

 The invention relates to the field of biotechnology and relates to a new strain of bacteria Escherichia coli, which can be used to obtain a restriction endonuclease (restrictase) that recognizes and cleaves the nucleotide sequence: 5'-GGCTCTCN! 3 '; 3'-CCAGAGNNNNN! 5 'as part of double-stranded DNA. The restriction enzyme Eco71kI produced by the proposed strain is an isoshizomer of the well-known restriction enzyme Eco31I and can be used in genetic engineering studies both in experiments on the construction of recombinant DNA in vitro and in studying the primary structure of DNA.

 Closest to the proposed strain is a strain of Escherichia coli RFL31, producing the restriction enzyme Eco311 [1] The enzyme yield is not given.

 The problem to which the invention is directed is to obtain a strain producing a restriction enzyme of a given specificity with a high yield, rapidly growing on ordinary nutrient media.

 The proposed strain Escherichia coli 71k was obtained as a result of a targeted search for strains of restriction enzyme producers among clinical isolators (Kiev) using our developed method for testing activity in extracts obtained from individual colonies grown on an agar medium, followed by electrophoresis of DNA cleavage products in agarose gel. The content of restriction enzyme Eco71kI in the proposed strain of 200,000 units / g of crude cell biomass.

 The strain is deposited in the All-Union Collection of Microorganisms at the IBPM RAS and is stored in it under the number VKM V-2006D.

 The strain Escherichia coli VKM V-2006D has the following characteristics.

 Cultural and morphological characters.

 Cells are straight rod-shaped, with a size of 0.7 0.8 μm across and 2 3, sometimes 5 μm, in length, mobile, gram-negative, with lateral flagella. They are located singly, in pairs or chains, not spore-bearing.

 The strain grows well on ordinary nutrient media (MPA, MPB, LB broth and LB agar). When growing on meat peptone agar or LB agar, the colonies are smooth, round, pressed, shiny, cloudy. When growing in liquid media: in the BCH-broth or LB-broth form a uniform intense turbidity.

 Physiological and biochemical characteristics.

Cells grow in the range from 4 to 50 ^o C, the optimum growth temperature of 37 ^o C, the optimum pH of 6.8 7.4.

 As a carbon source, many carbohydrates, alcohols, organic acids are used, in particular: alpha-glucose, alpha-fructose, galactose, arabinose, lactose, trehalactose.

 As a source of nitrogen, both mineral salts in ammonium and nitrate forms, and in organic form in the form of peptone, amino acids, are used.

 Nitrates are reduced to nitrites.

 Gelatin is not liquefied.

 Indole does not form.

 Genetic traits: Cells are resistant to tetracycline (20 μg / ml).

The storage conditions of the strain:
The bacterial strain Escherichia coli VKM V-2006D is stored on plates and jambs. Reseeding on fresh media once a month. It can be stored for at least a year in 15% glycerol at from -20 to -70 ^o C, in a freeze-dried state with sugar-gelatin agar, or in semi-liquid agarized LB medium under liquid paraffin.

 Example 1. Testing of restriction enzymes.

To search for restriction enzymes, the express method was used, which is a modification of the method of Belavin et al. [2] For this, one or two large colonies (about 5 • 10 ⁶ 1 • 10 ⁷ cells) are suspended in 20 μl of buffer A (20 mm Tris-HCl, pH 7.6, 12.5 mm EDTA, 100 mm NaCl, 10 mm 2-mercaptoethanol) containing 10 mg / ml lysozyme and incubated at room temperature (18 ^o C). After 30 min, an equal volume of buffer B (20 mM Tris-HCl, pH 7.6, 2% Triton X-100, 10 mM 2-mercaptoethanol) was added, and incubated for another 60 min at 4 ^° C. Cell suspension (1 and 4 μl) was mixed with 2 μg of DNA of phage ⌀ 80 vir in 40 μl of buffer C (10 mM Tris-HCl, pH 7.6, 10 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 50 mM NaCl). Hydrolysis is carried out at 37 ^o C for 10 minutes In addition, 4 μl of the cell suspension is further incubated in 40 μl of buffer C without substrate in order to verify the possible presence of extrachromosomal DNA in the solution. The reaction is stopped by extraction with an equal volume of water-saturated phenol. For electrophoretic analysis using 20 μl of hydrolyzate.

 Example 2. Obtaining and purification of restrictase.

The E. coli 71k cell culture is grown in 1 L of LB-broth (5 g of yeast extract, 10 g of NaCl, 10 g of bactotryptone per 1 l of water) at 37 degrees C to a titer of 4 • 10 ⁹ cells / ml. Cells are pelleted by centrifugation (6000 rpm for 15 minutes). 1 g of crude biomass is suspended in 5 ml of buffer containing 10 mM Tris HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA 10 mM 2-mercaptoethanol. Cells are destroyed by ultrasound, the extract is obtained by centrifugation at 2 ^o C (48000g).

To determine the activity of the restriction enzyme Eco71kI, serial dilutions of the supernatant are prepared: 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 in buffer (10 mM Tris HCl pH 7.5, 50 mM NaCl, 10 mM EDTA, 10 mM 2-mercaptoethanol). In an incubation mixture (9 μl) containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM MgCl ₂ , 1 mM EDTA, 10 mM DTE, 1 μg lambda phage DNA, add 1 μl of the appropriate dilution of the extract and incubate at 37 ^o C for 1 h. The results of hydrolysis are checked by electrophoresis in 0.8% agarose gel.

 The amount of enzyme required for complete specific cleavage of 1 μg lambda phage DNA for 1 hour is taken per unit of activity.

 The level of activity of the restriction enzyme Eco71kI, determined in the cell-free extract of the strain E. coli 71k, is about 200,000 units. in terms of 1 g of raw biomass.

 The restriction enzyme Eco71kI can be purified by fractionation of the cell extract sequentially on columns with DEAE cellulose (DE-52 Whatman) and phosphocellulose P-11 (Whatman), followed by chromatography on hydroxylapatite, carboxymethyl cellulose (CM-32 Whatman) and DNA agarose. The enzyme preparation is finally concentrated during dialysis against the buffer: 15 mM Tris-HCl, pH 7.5; 150 mM NaCl; 7 mM 2-mercaptoethanol; 1mM EDTA, 50% glycerin. The yield of purified enzyme is 5000 units per 1 g of crude biomass.

The purified enzyme is stable at -20 ^o C for six months. Restriction enzyme is suitable for DNA structure analysis and molecular cloning.

 Example 3. Determination of the nucleotide sequence recognized by the restriction enzyme Eco71kI and the place of DNA hydrolysis.

To determine the recognition sequence for restrictase, the lengths of fragments obtained experimentally by hydrolysis of phage lambda restriction enzyme DNA are compared with the lengths of fragments calculated on a computer using appropriate programs for different nucleotide sequences. Only one sequence was detected:
5'-GGTCTCN! -3 '
3'-CCAGAGNNNNN! -5 ',
for which the calculated values of the fragments coincided with the experimental ones and were localized in the following regions of the primary nucleotide sequence of lambda phage DNA: 11424, 42715.

 To confirm the obtained data, the recognition sites are mapped with the restriction enzyme Eco71kI on lambda phage DNA using co-hydrolysis with restriction enzymes: EcoRI and Eco71kI, BamHI and Eco71kI, CfrBI and Eco71kI.

Phage o 80 DNA was used to determine the site of DNA hydrolysis with Eco71kI restriction enzyme. DNA fragments obtained during hydrolysis are dephosphorylated by calf intestine phosphatase, inactivated by heating samples at 65 ^° C for 15 min, mixed with DNA ligase and in the presence of 0.5 mM ATP, carry out the ligation reaction. In a separate sample, the obtained fragments are mixed with DNA fragments of phage o 80 obtained by hydrolysis of Eco31I, not treated with phosphatase. Comparison of the electrophoretic profiles of DNA fragments after treatment with DNA ligase allows us to conclude that the hydrolysis site for the restriction enzymes Eco31I and Eco71kI is identical.The performed experiments show that the restriction enzyme Eco71kI is a true isoschisomer of the Eco31I restrictase.

The obtained restrictase Enco71kI is characterized by the following properties:
recognizes and specifically cleaves the nucleotide sequence in the DNA molecule 6 5'-GGTCTCN! -3 ';3'-CCAGAGNNNNN! -5 ';
the optimal pH for the action of restriction enzyme is 7.8 to 8.0;
optimal temperature action 37 ^o C.

NaCl 50 mM
BSA 100 mcg / ml
Thus, the obtained E. coli strain VKM B-2006D, as a producer of specific endonuclease Eco71kI, provides a high level of enzyme content of at least 200,000 units / g of crude biomass of cells, which is sufficient to obtain restriction enzyme in preparative quantities. The yield of purified enzyme is 5000 units / 1 g of cell biomass.

Claims (1)

 The bacterial strain Escherichia coli VKM B-2006D producer of restriction endonuclease Eco 71 k1.