RU2188234C2 - Method of preparing recombinant thymidine phosphorylase, recombinant plasmid dna pertpho1 and strain escherichia coli bl21(de3)/pertpho1 as producer of thymidine phosphorylase - Google Patents

Abstract

FIELD: biotechnology, biochemistry, genetic and protein engineering. SUBSTANCE: recombinant plasmid DNA pERTPHO1 encoding amino acid sequence of E. coli thymidine phosphorylase comprises Nde I/Sal I fragment of plasmid DNA pET20b(+) containing promoter and terminator of RNA-polymerase T7, enhancer of translation of gene 10 of phase T7, gene of β-lactamase and Nde I/Sal I-fragment of DNA containing a gene sequence encoding Escherichia coli thymidine phosphorylase adapted to these sites. The strain-producer of E. coli BL21(DE3)/pERTPHO1 is obtained by transformation of competent cells E. coli BL21(DE3) with plasmid pERTPHO1. The strain-producer E. coli BL21(DE3)/pERTPHO1 is grown on rich medium (YT-, LB-broth and others) (or induced with isopropylthio-β-D-galactoside and grown again) up to the attainment of maximal density of bacterial culture. Thymidine phosphorylase is isolated from cells of the producer after disruption of grown cells by one of conventional methods. Invention ensures to obtain recombinant thymidine phosphorylase with high yield and by simplified technology. EFFECT: improved method of preparing, increased yield. 3 cl, 1 dwg, 3 ex

Description

 The invention relates to biotechnology, in particular to genetic and protein engineering. It includes an in vitro recombinant plasmid DNA pERTPHO1 designed for biosynthesis of E. coli thymidine phosphorylase biosynthesis, E. coli strain BL21 (DE3) / pERTPHO1, a thymidine phosphorylase superproducer and a method for producing thymidine phosphorylase based on the aforementioned recombinant DNA recombinant.

 Escherichia coli thymidine phosphorylase (EC 2.4.2.4) catalyzes the catabolic phosphorolysis reaction of pyrimidine nucleosides in E. coli cells [1,2]. It is a protein with a molar mass of 47.3 kDa, functioning as a dimer with a mol. mass of 94.6 kDa [1]. The enzymatic activity of thymidine phosphorylase allows its use in the transglycosylation reaction in the synthesis of modified nucleosides, which are used in medicine as therapeutic drugs [3,4], or as a component of mixed antitumor drugs [5].

 For practical purposes, until recently, the enzymatic activity of whole bacterial cells of E. coli [6], as well as thymidine phosphorylase isolated from E. coli [7.8], or an enzyme in immobilized form [9], were mainly used.

 The cloning of the E. coli thymidine phosphorylase gene (deoA) in plasmid pKK-226 is known [7] with a relatively low biosynthesis efficiency (up to 0.4 g / l of culture).

 The closest to the claimed producer of thymidine phosphorylase E. coli with plasmid pUTPP is known, which allows obtaining high enzyme yield (up to 51% total protein content in cells) with IPTG-induced biosynthesis and a method for its isolation [8]. The present invention solves the problem of obtaining a highly productive recombinant bacterial strain-producer of the enzyme, allowing to obtain recombinant thymidine phosphorylase in high yield and by simplified technology.

 The problem is solved due to the fact that the producer strain obtained by transformation of Escherichia coli cells with plasmid DNA is cultured to the accumulation of recombinant thymidine phosphorylase in the amount of 60-70% of the total cell protein, the cells are destroyed in the buffer solution and the soluble fraction is separated. The thymidine phosphorylase content in this fraction is up to 80% of the total protein content, which can be used without further purification, either purified to a homogeneous state by gel chromatography, or its enzymatic activity can be increased under refolding conditions.

 A producer strain Escherichia coli BL21 (DE3) containing plasmid DNA pERTPHO1, a superproducer of thymidine phosphorylase, is used.

Use recombinant plasmid DNA pERTPHO1
- the coding amino acid sequence of thymidine phosphorylase E. coli;
- having a molecular weight of 2.98 MDa;
- consisting of:
An NdeI / SalI DNA fragment of plasmid pET20b (+) containing a promoter and terminator of transcription of T7 RNA polymerase, a translation enhancer of T7 phage 10 gene, β-lactamase gene, and an NdeI / SalI DNA fragment containing a gene sequence adapted to these sites thymidine phosphorylases of Escherichia coli;
- containing:
as a genetic marker, the β-lactamase gene determining the resistance of penicillin antibiotics transformed with plasmid pERTPHO1 to E. coli; unique recognition sites for restriction endonucleases located to the right of the NdeI site: XbaI - 38 bp, BglII - 96 bp, PvuII - 741 bp, PstI - 2288 bp, PvuI - 2413 p . o., SalI - 4918 bp

 Use the producer strain Escherichia coli BL21 (DE3) containing the recombinant plasmid DNA pERTPHO1, a producer of thymidine phosphorylase.

 The present invention allows to obtain recombinant thymidine phosphorylase by simple technology and in high yield.

 The design of recombinant plasmid DNA pERTPHO1 provides a high level of expression of the thymidine phosphorylase gene cloned therein.

 A chemical approach was used to construct the plasmid, which allows the use of optimal regulatory elements that control its expression for the expression of the cloned structural gene.

 The source of the structural gene for thymidine phosphorylase is E. coli chromosomal DNA. The recombinant gene is isolated by PCR with synthetic oligonucleotide primers and then cloned into the vector plasmid pET-20b.

The proposed producer strain Escherichia coli BL21 (DE3) / pERTPHO1 is characterized by the following features:
Morphological signs. The cells are rod-shaped, gram-negative, non-spore.

 Cultural signs. Cells grow well on simple nutrient media. When growing on Difco agar, the colonies are round, smooth, cloudy, shiny gray, the edge is even. When growing on liquid media (on a minimal medium with glucose or YT-broth) they form an intense smooth turbidity.

Physico-biological signs. Cells grow at a temperature of from 4 ^o C to 40 ^o With an optimum pH of 6.8 to 7.5. As a source of nitrogen, both mineral salts in ammonium form and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. are used. Amino acids, glycerin, carbohydrates are used as a carbon source.

 Antibiotic resistance. Cells are resistant to penicillin antibiotics (up to 500 μg / ml).

 The strain producing E. coli BL21 (DE3) / pERTPHO1 differs from the recipient strain E. coli BL21 (DE3) only in the presence of recombinant plasmid DNA pERTPHO1, which gives it resistance to penicillin antibiotics.

 Producer strains are obtained by transformation of competent E. coli BL21 (DE3) cells with the corresponding recombinant plasmid DNA.

 E. coli cells BL21 (DE3) / pERTPHO1 are a superproducer of thymidine phosphorylase. With the induction of isopropylthio-β-D-galactoside, as well as without induction, an effective biosynthesis of thymidine phosphorylase occurs, which accumulates in the cells in an amount of more than 60% of the total bacterial protein.

 The producer strain was deposited in the All-Russian Collection of Industrial Microorganisms, VKPM B-7976 dated May 25, 2000.

 The invention is as follows. Recombinant plasmid DNA pERTPHO1 was constructed, for which the thymidine phosphorylase gene was isolated from E. coli chromosomal DNA using PCR with synthetic oligonucleotide primers (drawing) containing NdeI restriction enzyme sites (N-terminus of the gene, primer A1) and SalI (C-terminus of the gene , primer B1), the obtained DNA was digested with the corresponding restriction enzymes and then ligated with the pET-20b vector plasmid cleaved at the same sites.

The competent E. coli BL21 (DE3) cells are transformed with the ligase mixture and plated on YT agar containing 50 μg / ml ampicillin or another penicillin antibiotic. The resulting clones were analyzed by hybridization with ³² P-labeled oligonucleotides A1 and B1 (drawing), and plasmid DNA was isolated from hybridizing clones, which was subjected to restriction analysis using restriction enzymes NdeI and SalI. The E. coli producing strain BL21 (DE3) / pERTPHO1 is grown in a rich medium (YT-, LB-broth, etc.) (or isopropylthio-β-D-galactoside is induced and grown again) until the culture density is reached.

Isolation of thymidine phosphorylase from producer cells involves the following steps:
- destruction of the grown cells using ultrasound;
- separation of the soluble fraction by centrifugation; the content of thymidine phosphorylase in this fraction is up to 80% of the total protein content, and the enzyme can be used without further purification;
- from the soluble fraction, the target protein can be purified to a homogeneous state by standard methods.

 The drawing shows the structure of the thymidine phosphorylase gene and synthetic primers used to isolate the gene by PCR and select clones by hybridization.

 The invention is illustrated by the following examples.

Example 1. Construction of recombinant plasmid DNA pERTPHO1
Chemical synthesis of oligonucleotides is carried out by the solid-state phosphoamidite method on an ASM-102U DNA synthesizer (BIOSSET, Novosibirsk) with the oligonucleotide chain being expanded from the 3'-end to the 5'-end using protected phosphamidites - 5'-dimethoxytrityl-N-acyl-2 '-deoxynucleoside-3'-O- (β-cyanethyl-diisopropylamino) -phosphites activated by tetrazole. The synthesis is carried out on a scale of 0.5-0.7 μmol, using porous glass (pore size 500 A) as a support, to which the first nucleoside unit (load 20-30 μmol / g) is connected via a 3'-succinate bond. The synthetic cycle described in [10] is used.

To prepare the vector of the plasmid pET-20b DNA (3 μg, 1 pmol), it is treated with 40 μl of Buffer R (10 mM Tris-HCl, pH 8.5, 10 mM MgCl ₂ , 100 mM KCl, 0.1 mg / ml BSA) restriction enzyme NdeI (10 units), and then in 40 μl of buffer O (50 mm Tris-HCl, pH 7.5, 10 mm MgCl ₂ , 100 mm NaCl 1 mm DTT, 0.1 mg / ml BSA) restriction enzyme SalI (10 units) for 1 hour at 37 ^° C. A vector fragment of 3.6 kbp. after electrophoresis in a 1% agarose gel, it is electrophoretically transferred to a layer of DEAE paper, then 1M NaCl is eluted and DNA is precipitated from the solution with ethanol.

To prepare the thymidine phosphorylase gene fragment, PCR amplification is carried out using E. coli chromosomal DNA (0.01 μg in the sample) as a template, and synthetic oligonucleotides A1 and B1 (60 pmol each) as primers. PCR is carried out in a DNA amplifier, in a buffer solution containing each of the four dNTPs at a concentration of 0.5 mM and 5 units. Act. Taq DNA polymerase, in the following mode: denaturation - 1 min at 94 ^° C, annealing - 30 sec at 60 ^° C, elongation - 40 sec at 72 ^° C, 30 PCR cycles. After this, the reaction mixture was deproteinized with chloroform, evaporated to dryness, the residue was dissolved in 20 μl of water, then digested with the same restriction enzymes used in the preparation of the vector, and the target fragment was isolated from the agarose gel.

The resulting synthetic fragment with the thymidine phosphorylase gene in an amount of 2 pmol is added to a solution of 1 μg of the above vector fragment in 10 μl of buffer (20 mm Tris-HCl, pH 7.56, 10 mm MgCl ₂ , 0.2 mm rATP, 10 mm dithiothreitis) and are ligated with 10 units of act. T4-DNA ligase for 12 hours at 10 ^o C.

An aliquot of the reaction mixture is used to transform competent E. coli BL21 cells (DE3). Transformants are plated on YT agar plates containing 50 μg / ml ampicillin. Recombinants are screened by in situ hybridization of the colonies with ³² P-labeled oligonucleotide A1 (Figure). The pERTPHO1 plasmid DNA was isolated from hybridizing clones and analyzed using NdeI and SalI endonucleases.

 Example 2. Obtaining a strain of E. coli BL21 (DE3) / pERTPHO1 (VKPM B-7976) - producer of thymidine phosphorylase and determination of its productivity.

 E. coli BL21 (DE3) cells carrying the plasmid pERTPHO1, the structure of which is confirmed by the analysis data (see Example 1), are a superproducer of thymidine phosphorylase.

The producer strain E. coli BL21 (DE3) / pERTPHO1 was grown at 37 ^{° C.} in 100 ml of YT broth (pH 7.0) with 50 μg / ml ampicillin for 2 hours on a shaker at a speed of 190 rpm until turbidity A ₅₅₀ 0.7-0.8, isopropylthio-β-D-galactoside is added to a concentration of 0.2 mM and the process is continued for another 6 hours. Each hour, a 2 ml sample is taken, A ₅₅₀ is determined and the amount of culture corresponding to 1 ml with A ₅₅₀ 1.0, centrifuged for 5 minutes at 6000 rpm. Precipitated cells in 100 μl of lysing buffer with bromphenol blue dye are sonicated for 20 seconds, heated for 3 minutes at 100 ^° C and 1 μl samples are used for electrophoresis in 15% SDS-PAGE. The gel was stained with Coomassie R-250 according to a standard procedure and scanned to determine the relative amount of protein in the target protein band.

 Example 3. Obtaining thymidine phosphorylase.

Wet cells (10 g) are suspended in 20 ml of buffer (30 mM Na-phosphate, pH 7.0, 5% glycerol) and destroyed by ultrasound using an ultrasonic disintegrator Sonifier 240 (Branson) (3 pulses of 20 sec at A 2.0 and 0 ^o C). The homogenate is centrifuged for 10 min at 10,000 g and the obtained supernatant with an enzyme content of up to 80% of the total protein is used for the transglycosylation reaction.

Sources of information
1. O'Donovan GA, Neuhard Y. // Bacteriol. Revs., 1970, v. 34, p. 278.

 2. Mironov A.S., Smirnov Yu.V., Sukhodolets V.V. // Genetics, 1975, v. 11, p. 97.

 3. Chae W. G., Cauchon N. S., Kozlowski J. F., Chang C. // J. Org. Chem., 1992, v. 57, p. 1002-1005.

 4. Schinazi R.F., Peck A., Sommadossi J.P. // Biochem. Pharmacol., 1992, v. 44, p. 199-204.

 5. WO Pat. 9308273, 04.29.93.

 6. Khachatourians G. G., Brosseau J. D., Child J. J. // Biotechnol. Lett., 1982, v. 4, p. 735-740.

 7. Barbac. III, C.F., Wong C.-H. // Bioorgan.Chem., 1991, v.l9, p. 261-269.

 8. Veiko V.P., Siprashvili 3.3., Ratmanova K.I., Gulko LB, Andryukhina R.V., Debabov V.G. // Biotechnology, 1994, v.4, p.2-4 .

 9. Gaevaya L.V., Zhukov V.N.//App. Biochem. Microbiol., 1987, v. 23, p. 309-316.

 10. Atkinson, T., Smith M. // in: Oligonucleotide synthesis; a practical approach. 1984. Ed. Gait M.J. p. 35-81. IRL Press, Oxford.

Claims (3)

 1. A method for producing recombinant thymidine phosphorylase, comprising cultivating a producer strain obtained by transforming Escherichia coli cells with plasmid DNA followed by cell disruption in a buffer solution, characterized in that recombinant plasmid pERTPHO1 is used as plasmid DNA, and a producer strain is used strain Escherichia coli BL21 (DE3) / pERTPHO1, which is cultivated in a rich medium, the destruction of the cells is carried out by ultrasound, then the soluble fraction is separated.  2. Recombinant plasmid DNA pERTPHO1, encoding the amino acid sequence of Escherichia coli thymidine phosphorylase, having a molecular weight of 2.98 MDa, consisting of an NdeI / SalI DNA fragment of plasmid pET20b (+), containing a promoter and T7 RNA polymerase transcription terminator, enhancer translation of phage T7 gene 10, β-lactamase gene, NdeI / SalI DNA fragment containing the Escherichia coli thymidine phosphorylase gene sequence adapted to these sites, containing β-lactamase gene determining the stability of penicillin antibiotics, unique recognition sites of restriction endonucleases located at the following distance to the right from the NdeI site: XbaI - 38 bp, BglII - 96 bp, PvuII - 741 bp, PstI - 2288 bp, PvuI - 2413 bp, SalI - 4918 bp  3. The strain Escherichia coli BL21 (DE3) (VKPM B-7976) containing the recombinant plasmid DNA pERTPHO1 according to claim 2, the producer of thymidine phosphorylase.

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