RU2054037C1 - Recombinant plasmid dna peco 29ki encoding synthesis of restriction endonuclease eco 29ki and strain escherichia coli - a producer of restriction endonuclease eco 29ki - Google Patents

Abstract

FIELD: genetic engineering. SUBSTANCE: restrictase Eco 29KI is isoschizomer of restrictase SacII. The new recombinant plasmid pECO 29KI (size is 6.6 thousand nucleotide pairs) is obtained and it has the genes of restriction-modification system Eco 29KI, consists of ClaI-fragment od DNA-vector PUC 129 and two ClaI-fragments of the natural plasmid p29k, has also four recognition sizes by restrictase HindIII, three recognition sites of restrictase, two sites for XhoI, and one site for restrictases: SalI, BglII, Bam HI, resistance dominant to ampicillin. Obtained plasmid provides the increased synthesis of restrictase Eco 29KI. Proposed strain of Escherichia coli BKM CR-369D prepared by transformation of plasmid DNA pECO 29KI into the strain E. coli Z85 provides high level of enzyme content, 100000000 U/1 g wet biomass. The yield of purified enzyme is 500000 U/g biomass. EFFECT: increased yield of enzyme. 3 cl

Description

 The invention relates to biotechnology and is a recombinant plasmid DNA that determines the synthesis of restriction endonuclease Eco29kI and a bacterial strain Escherichia coli, which can be used to obtain a restriction endonuclease (restriction enzyme) that recognizes and cleaves the nucleotide sequence 5'-CCGCiGG-3 'in double-stranded DNA . The restriction enzyme Eco29kI produced by the proposed strain is an isoshizomer of the known restriction enzyme SacII and can be used in genetic engineering studies, both in experiments on the construction of recombinant DNAs in vitro, and in studying the primary structure of DNA.

 The known restriction enzyme SacII produced by the strain of bacteria Streptomyces achromogenes ATCC 12767, which recognizes and cleaves a portion of DNA 5'-CCgCIGG-3 '. The content of restriction enzyme in the biomass is not indicated.

 Also known is the SacII restriction enzyme isoschizomer isolated from the Gluconobacter albidus IFO 3262 strain, the preparation of which is described in US Pat. No. 4,668,631. The yield of purified enzyme is 375 units per 1 g of crude cell biomass.

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

 The essence of the proposed inventions is that a recombinant plasmid pECO29kI with the genes of the restriction-modification system Eco29kI, which causes the synthesis of restriction enzyme Eco29kI, and a bacterial strain containing this recombinant DNA producer restriction enzyme Eco29kI, were obtained.

 The resulting recombinant plasmid pECO29kI size of 6.6 TPN encoding the restriction endonuclease Eco29kI, is non-conjugative, compatible with ColE1-type multicopy plasmids, and consists of the following elements: two ClaI DNA fragments of the natural plasmid p29k in size: 1.95 kb each containing the restriction-modification genes of Eco29kI and the ClaI DNA fragment of pUC129 2.7 kb in size. The plasmid contains four recognition sites with the restriction enzyme HingIII located at a distance of 0.68 kb. 1.4 kb 1.75 kb and 2.8 kb three recognition sites for R. ClaI located at a distance of 1.95 kb 1.95 kb 2.7 kb two recognition sites for R.XhoI located at a distance of 1.72 kb 4.9 kb and one recognition site by restriction enzymes SalI, BgIII, BamHI; bla-gene, responsible for the synthesis of beta-lactamase, which provides cell resistance to ampicillin; eco29kIR gene encoding the restriction endonuclease Eco29kI, providing phage restriction of ⌀ 80 vir; eco29kIM gene encoding Eco29kI methylase, which provides modification of the CCGCGC nucleotide sequence and protects DNA from restriction enzyme digestion with Eco29kI; ori-replicon of plasmid pMB1.

 A method for constructing a plasmid is that the plasmid DNA of vector pUC129 is hydrolyzed with a ClaI restriction endonuclease and DNA ligase is coupled to DNA of a natural plasmid p29k digested with the same restriction enzyme under conditions of incomplete hydrolysis. A mixture of the combined fragments is transformed into Escherichia coli K802 cells containing the natural plasmid p29k. Transformants are plated on medium with ampicillin and extrachromosomal DNA is isolated from the grown clones, which transform competent E. coli K802 cells. Transformants were selected on an agar medium containing ampicillin and bacteriophage lambda vir, and plasmid DNA designated pECO29kI was isolated from the clones obtained on this medium.

 The proposed producer strain restriction endonuclease Eco29kI is obtained by transformation of plasmid DNA pECO29kI into Escherichia coli strain Z85. In this strain, plasmid DNA pECO29kI is stable and provides increased enzyme synthesis. The content of restriction enzyme Eco29kI in the proposed strain of 10 million units / gram of crude cell biomass.

 The strain is deposited in the All-Union Collection of Microorganisms at the Institute of Fundamental Physics of the Russian Academy of Sciences and is stored in it under the number VKM CR-369D.

 The strain Escherichia coli BKM CR-369D has the following characteristics. Cultural and morphological characters. The cells are straight, rod-shaped, with a size of 0.7-0.8 microns across and 2-3, sometimes 5 microns, 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 growing in a range of 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, 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: genotype Δ (lac pro), thi, strA, supE, endA, sbcB, hsdR-, F'traD36, proAB, lac1q, ZЛМ15, recA: Tn10. The strain is resistant to ampicillin (50 μg / ml), due to the presence of the beta-lactamase gene in the pECO29kI DNA, which ensures the resistance of cells with the plasmid to ampicillin and tetracycline (20 μg / ml), due to the presence of Tn10 transposon in strain Z85.

The storage conditions of the strain:
The bacterial strain Escherichia coli BKM CR-369D is stored on plates and jambs. Reseeding on fresh media once a month. 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. Construction of plasmids pECO29kI.

Ekstrahromosomolnuyu DNA, in particular, p29k plasmid was extracted from a natural strain of E.coli 29k, gdirolizuyut restriction enzyme ClaI in incomplete hydrolysis conditions at 37 ^{° C} for 15 min in buffer containing 10 mM Tris HCl, pH7,5, 50 mM NaCl, 10 mM MgC12, 1 mM dithiothreitol. The vector plasmid DNA pUC129 was hydrolyzed with the restriction enzyme ClaI for one hour in the buffer indicated above. Enzymes are inactivated by extraction of DNA with water-saturated phenol and, after ethanol reprecipitation, 2 kg of ClaI-DNA hydrolyzate from E. coli 29k is mixed with 0.2 μg of pUC129 ClaI-hydrolyzate, ATP is added to 5 mM and the reaction is carried out in the presence of 0.1 units. DNA ligase at 10 14 ^{° C} for 16 hours in buffer 66 mM tris-HCl pH 7,5, 50 mM NaCl, 10 mM MgC12, 1 mM dithiothreitol. The lysaga is inactivated by heating at 65 ° C. ^for 10 minutes and the incubation mixture is used to transform competent E. coli K802 cells containing the natural plasmid p29k.

 From transformants grown on a selective agar medium with ampicillin (50 μg / ml), total extrachromosomal DNA is isolated and used to transform E.coli K802 cells. Among the transformants grown on medium with ampicillin and with the bacteriophage vir lambda in the amount of 100 phage particles per bacterial cell, clones containing the Eco29kI restriction-modification system were selected. PECO29kI plasmid DNA was isolated from the obtained transformants and restriction analysis was performed using restriction endonucleases: HindIII, EcoRI, AvaI, SmaI, ClaI, XhoI, KpnI, SalI using the appropriate incubation buffers.

 PRI me R 2. Obtaining a strain of Escherichia coli Z85 (pECO29kI).

 Plasmid pECO29kI transform competent E. coli Z85 cells. Among the transformants grown on medium with ampicillin (50 μg / ml) and bacteriophage vir lambda in the amount of 100 phage particles per cell, clones containing the Eco29kI restriction-modification system were selected and one of them was used as the Eco29I restriction enzyme producer strain.

 PRI me R 3. Obtaining and purification of restrictase.

A cell culture of E. coli Z85 (pECO29kI) is grown in 1 L of LB broth (5 g of yeast extract, 10 g of NaCl, 10 g of bactotrypton per 1 l of water) at 37 ° C to a titer of 2x10 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, 1 mM EDTA, 10 mM 2-mercaptoethanol. Cells are destroyed by ultrasound, the resulting extract is centrifuged at 48000 g at 2 ^about C.

 To determine the activity of Eco29kI 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, 1 mM EDTA, 10 mM 2-mercaptoethanol). To the incubation mixture (9 μl) containing 10 mM Tris HCl, pH 7.5, 50 mM NaCl, 75 mM KCl, 10 mM MgC12, 1 mM EDTA, 10 mM DTE, 1 μg lambda phage DNA, add 1 μl of the appropriate dilution extract and incubated at 37 deg. C for 1 h. The hydrolysis results are checked by electrophoresis on a 1.2% 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 Eco29kI rextractase, determined in the cell-free extract of the E. coli strain Z85 (pECO29kI), is about 10,000,000 units in terms of 1 g of raw biomass.

 The restriction enzyme Eco29kI can be purified by fractionation of the cell extract sequentially on columns with DEAE cellulose (DE-B2 Whatman), followed by chromatography on phosphocellulose (P11, Whatman), hydroxylapatite and heparin agarose when fractionated with ammonium sulfate after chromatography on phosphocellulose. The enzyme preparation is finally concentrated during dialysis against 50% glycerol. The yield of purified enzyme is 500,000 units / g of cell biomass.

The purified enzyme is stable at ^-20 ° C for six months. At 20 ^{° C} Eco29kI restriction enzyme retains 95% of its activity for 14 days at ³⁷ C 50% for 14 days, at 65 ^C 95% for 60 min. Restriction enzyme is suitable for DNA structure analysis and molecular cloning.

 Example 4. Determination of the nucleotide sequence recognized by the restriction enzyme Eco29kI and the site of DNA hydrolysis.

 To determine the recognition sequence of the restriction enzyme, the fragment lengths obtained experimentally by hydrolysis of lambda phage DNA with the restriction enzyme Eco29kI are compared with the fragment lengths calculated on a computer using appropriate programs for different nucleotide sequences. Only one 5'-CCGC! GG-3 'sequence was found, for which the calculated fragment sizes coincided with the experimental ones and were localized in the following regions of the primary nucleotide sequence of lambda phage DNA (bp): 20323, 20533, 21609, 40389.

 To confirm the obtained data, mapping of recognition sites by restriction enzyme Eco29kI onto lambda phage DNA is carried out using joint hydrolysis with restriction enzyme NruI.

 PUC128 DNA was used to determine the site of DNA hydrolysis by restriction enzyme Eco29kI. After annealing with a synthetic primer (17-mer) and a polymerization reaction, the matrix is treated with an Eco29kI restriction endonuclease. A portion of the sample is polymerized a second time and both of the resulting preparations are tested under the conditions proposed by Simcon et al. Four standard sequencing reactions are carried out according to the protocol of Sanger et al.

Our experiments allow us to make an unambiguous conclusion that the restriction enzyme Eco29kI is a true isoschisomer of the restriction enzyme SacII and recognizes the nucleotide sequence
5'-CCGC! GG-3 '
In addition, the obtained restriction enzyme Eco29kI is characterized by the following optimal reaction conditions:
optimal pH for the action of restriction enzyme 7.2-8.5
the optimum temperature is 37 ^o C.

NaCl 20-120 mm
Thus, the recombinant plasmid pECO29kI containing the genes of the Eco29kI restriction-modification system was obtained. This plasmid provides a high level of synthesis of restriction endonuclease Eco29kI.

 The proposed strain of E.coli BKM CR-369D, as a producer of specific endonuclease Eco29kI, provides a high level of enzyme content of at least 10,000,000 units per 1 g of crude biomass of cells, which is sufficient to obtain restriction enzyme in preparative quantities. The yield of purified enzyme is at least 500,000 units / g of cell biomass, which is 1300 times higher than in the case of the prototype.

Claims (2)

 1. Recombinant plasmid DNA pEco29kI - determining the synthesis of 6,6 kb Eco29kI restriction endonuclease, containing two ClaI DNA fragments of the natural plasmid p29k 1,95 kb each, the restriction system genome - Eco29kI modifications; The ClaI DNA fragment of the vector puC129 measuring 2.7 bp; restriction sites: four recognition sites of the restriction enzyme Hind III, located at a distance of 0.68; 1.4; 1.75 and 2.8 kb; three recognition sites for ClaI restriction enzyme; - two recognition sites for the restriction enzyme XhoI, located at a distance of 1.72 and 4.9 TPK; - Unique recognition sites for restriction enzymes: Sal I, Bgl II, Bam H I; - genetic markers: - bla-gene responsible for the synthesis of beta-lactamase, which provides resistance of cells to ampicillin, eco29kIR-gene encoding the restriction endonuclease Eco29kI, providing phage restriction with a diameter of 80 vir; - eco29kIM gene encoding Eco29kI methylase, which provides modification of the CCSGG nucleotide sequence and protects DNA from restriction enzyme digestion with Eco29kI; - ori-reclipcon plasmids pMBI.  2. The bacterial strain Escherichia coli VKM CR-369D - producer of restriction endonuclease Eco29kI.