RU2044053C1 - Strain of bacterium escherichia coli - a producer of restriction endonuclease eco27k1 - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: product: restriction endonuclease Eco27K1 recognizing and splitting the nucleotide sequence 5′-C!PyCGPuG-3′. Restrictase produced by proposed strain is an isoschizomer of restrictase AvaI and can be used in genetic engineering investigations. Proposed strain Escherichia coli BKM B-2005D is isolated from the clinical isolates and provides the high level of enzyme content 500000 U/g crude matter of cell biomass, not less. The yield of purified enzyme is 30000 U/g cell biomass. EFFECT: increased activity of enzyme proposed.

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 restriction endonuclease (restrictase), which recognizes and cleaves the sequence of nucleotides 5'-C! PyCGPuG-3 'as part of double-stranded DNA. The restriction enzyme Eco27kI produced by the proposed strain is an isoshizomer of the known restriction enzyme AvaI 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.

 Known isoshizomers of AvaI restriction enzymes isolated from various strains: Agmenellum quadruplicatum (PR-), Nostoc PCC7524.

 Closest to the proposed strain is a strain of Escherichia coli RFL88 [1] producing the restriction enzyme Eco881, which is the true AvaI restriction enzyme isoschisomer and recognizes and cleaves the 5'-C! PyCGPuG-3 'DNA nucleotide sequence [3] The yield of the Eco881 enzyme is not indicated.

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

 The proposed strain Escherichia coli 27k was obtained as a result of a targeted search for strains producing restriction enzymes among clinical isolates (Kiev) using the developed method for testing activity in extracts obtained from individual colonies grown on an agar medium followed by electrophoresis of the products of DNA cleavage in agarose gel. The content of restriction enzyme Eco27kI in the proposed strain of 500,000 units / per gram 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-2005D.

 The strain Escherichia coli VKM V-2005D has the following characteristics.

 Cultural and morphological characters.

 The cells are straight, rod-shaped, 0.7-0.8 microns in diameter and 2-3, sometimes 5 microns in length, motile, 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 growing in a range of from 4 ^to 50 ° C, optimum growth temperature 37 ^{° C,} optimum pH 6.8-7.4. As a carbon source, many carbohydrates, alcohols, organic acids are used, in particular: alpha-glucose, alpha-fructose, lagactose, arabinose, lactose, trehalactose. As a source of nitrogen, mineral salts are used in the ammonium and sodium forms, and in the organic form in the form of peptone, amino acids. Nitrates are reduced to nitrites. Gelatin is not liquefied. Indole does not form.

 Ginetic signs. Cells are resistant to ampicillin (Am-20 μg / ml), novobiocin (Nb 20 μg / ml), streptomycin (Sm 20 μg / ml).

The storage conditions of the strain:
The bacterial strain Escherichia coli VKM B-2005D is stored on plates and jambs. Reseeding on fresh media once a month. The strain can be stored for at least one year in 15% glycerol at -20-70 ^{° C,} in the freeze-dried state with a sugar-gelatin or agar agar semi-solid LB medium under mineral oil.

PRI me R 1. Testing restriction enzymes. To search for restriction enzymes, the express method was used, which is a modification of the Belavin et al. Method [3]. For this, one or two large colonies (about 5x10 ⁶ 1x10 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 ^about 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) and incubated another 60 minutes at 4 ^C. The cell suspension (1 and 4 ul ) mixed with 2 μg of DNA of phage with a diameter of 80 vir in 40 μl of buffer C (10 mm Tris-HCl, pH 7.6, 10 mm MgCl, 10 mm 2-mercaptoethanol, 50 mm NaCl). Hydrolysis is carried out at 37 ^about 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.

 PRI me R 2. Obtaining and purification of restrictase.

E.coli 27k cell culture was grown in 1 liter LB-broth (5 g yeast extract, 10 mg of NaCl, 10 g Bacto trypton 1 liter of water) at ³⁷ ° C to a titer of 4x10 ⁹ cells / ml. Cells are pelleted by centrifugation (6000 rpm, 15 min). 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 ^about C (48000g). To determine the activity of Eco27kI restriction enzyme, 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, 1 μg phage lambda phage, add 1 μl of the appropriate dilution of the extract and incubated at 37 ^about C for 1 h. The hydrolysis results are checked by electrophoresis in 0.8% agarose gel. The amount of enzyme necessary for complete specific cleavage of 1 μg of lambda phage DNA for 1 h is taken as a unit of activity.

The level of activity of the restriction enzyme Eco27kI, determined in the cell-free extract of E. coli strain 27k, is about 500,000 units in terms of 1 g of crude cell biomass. The restriction enzyme Eco27kI can be purified by fractionation of the cell extract sequentially on a DEAE cellulose column followed by chromatography on DEAE-Toeyperl, SP-Toeyperl, hydroxylapatite and heparinsepharose. The enzyme preparation is finally concentrated during dialysis against the buffer: 100 mM NaCl, 20 mM Tris-HCl, pH 7.6, 7 mM 2-mercaptoethanol, 50% glycerol. The yield of purified enzyme is: 30,000 units per g of raw biomass. The obtained restriction enzyme Eco27kI is characterized by the following properties: it recognizes and specifically cleaves the nucleotide sequence in the 5'-C DNA molecule! PyCGPuG-3 '. The purified enzyme at ^-20 ° C for six months retains 95% of its activity. At 0 ^о С the restriction enzyme Eco27kI retains 95% of its activity for 14 days, at +65 ^о С it loses 95% of its activity within 15 minutes. Restriction enzyme is suitable for DNA structure analysis and molecular cloning.

 PRI me R 3. Determination of the nucleotide sequence recognized by the restriction enzyme Eco27kI and the place of DNA hydrolysis. To determine the sequence of recognition by a restriction enzyme, a comparison is made of the lengths of fragments obtained experimentally by hydrolysis of DNA of phage lambda by restriction enzyme with the lengths of fragments calculated on the EPM using appropriate programs for different nucleotide sequences. Only one 5'-C! PyCGPuG-3 'sequence was found, for which the calculated fragment sizes coincided with the experimental ones and were located in the following regions of the primary nucleotide sequence of lambda phage DNA: 4721, 19398, 21000, 27888, 31618, 33499, 38215, 39889. To confirm the obtained data, mapping of recognition sites by restriction enzyme Eco27kI onto lambda phage DNA is performed using co-hydrolysis with restriction enzymes: EcoRI and Eco27kI, BamHI and Eco27kI, CfrBI and Eco27kI.

 The restriction enzyme Eco27kI was shown to hydrolyze this DNA in positions corresponding to the regions of hydrolysis by AvaI restrictase. M13tg 130 DNA was used to determine the site of DNA hydrolysis with Eco27kI restriction enzyme. After annealing with a universal synthetic primer and polymerization reaction, the matrix was treated with an Eco27kI restriction endonuclease. A part of the sample was polymerized a second time, and both obtained preparations were studied under the conditions proposed by Sanger [7] using an 8% denaturing polyacrylamide gel, which made it possible to determine the site of hydrolysis.

The experiments allow us to conclude that the restriction enzyme Eco27kI is a true isoschisomer of the AvaI restriction enzyme and recognizes the nucleotide sequence:
-5'-C! PyCGPuG-3 '
In addition, the obtained restriction enzyme Eco27kI is characterized by the following optimal reaction conditions: pH 7.5, 37 ^about C, 50 mm NaCl.

 Thus, the obtained strain of Escherichia coli VKM B-2005D, as a producer of specific endonuclease Eco27kI, provides a high level of enzyme content of at least 500,000 units per 1 g of crude biomass of cells, which is sufficient to obtain restriction enzymes in preparative quantities. The yield of purified enzyme is 30,000 units per g of biomass of cells.

Claims (1)

 The bacterial strain Escherichia coli VKM B-2005 producer of restriction endonuclease Eco 27K1.