RU2177998C2 - Method of preparing recombinant uridine phosphorylase, recombinant plasmid dna perurph01 and strain of escherichia coli bl 21(dez)/perurph01 for its realization - Google Patents

Abstract

FIELD: biotechnology, genetic and protein engineering, biochemistry. SUBSTANCE: recombinant plasmid DNA pERURPH01 encoding amino acid sequence of uridine phosphorylase of E. coli consists of DNA fragment Nde I/Sal I of plasmid pET20b(+) containing promoter and terminator of transcription of T7-RNA-polymerase, enhancer of translation of gene 10 phase T7, β- lactamase gene and DNA fragment Nde I/Sal I containing the sequence of gene encoding uridine phosphorylase of Escherichia coli adapted to this sites. The strain-producer is E. coli 21 (DEZ)/pERURPH01 obtained by transformation of E. coli cells by plasmid DNA pERURPH01 is cultured to accumulation of recombinant uridine phosphorylase in the amount 60-70% of total protein level. Cells are disrupted by ultrasonic oscillation in buffer solution and soluble fraction is separated. The strain-producer E. coli BL 21(DEZ)/pERURPH01 is cultured in rich medium (YT-, LB-broth and others) or induced with isopropylthio-β-D-galactoside and cultured again up to maximal culture density. Invention ensures to obtain uridine phosphorylase of E. coli with high yield (80% of total protein content). EFFECT: improved method of preparing, increased yield of enzyme, simplified technology. 4 cl, 3 ex

Description

 The invention relates to biotechnology, in particular to genetic and protein engineering. It includes an in vitro recombinant plasmid DNA pERURPHO1 designed for biosynthesis of E. coli uridine phosphorylase biosynthesis, E. coli strain BL21 (DE3) / pERURPHO1 - a superproducer of uridine phosphorylase and a method for producing uridine phosphorylase based on the above recombinant recombinant DNA transglycosylation reactions in the synthesis of nucleosides.

 Uridine phosphorylase of Escherichia coli (EC 2.4.2.3) is a protein with mol. weighing 27 Kda, functioning as a hexamer with a mol. weighing 162 Kda [3], and catalyzes the catabolic reaction of uridine phosphorolysis in E. coli cells [1,2], which allows its use in the transglycosylation reaction in the synthesis of modified nucleosides, which are used in medicine as therapeutic agents [4].

 For practical purposes, until recently, they used not only isolated uridine phosphorylase from E. coli, but mainly the enzymatic activity of whole bacterial E. coli cells, or a mixture of nucleoside phosphorylases from these cells without enrichment or separation [5-8].

 Closest to the application is a method for producing uridine phosphorylase from a producer strain E. coli obtained using microbiological selection methods [11]. Cloning of the E. coli uridine phosphorylase gene in plasmid pVMK27 [3] and plasmid pUUDP [10] has also been described, but data on the productivity of the recombinant enzyme producer strain have not been reported.

 The present invention solves the problem of obtaining a highly productive recombinant bacterial strain producing the enzyme, recombinant uridine 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 uridine 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 content of uridine phosphorylase in this fraction is up to 80% of the total protein content, and the enzyme can be used without further purification or purified to a homogeneous state by standard methods.

 Use the producer strain Escherichia coli BL21 (DE3) containing the plasmid DNA pERURPHO1 - superproducer of uridine phosphorylase.

Use recombinant plasmid DNA pERURPHO1:
- The coding amino acid sequence of E. coli uridine phosphorylase;
- having a molecular weight of 2.94 MDa;
- consisting of:
An NdeI / SalI DNA fragment of plasmid pET20b (+) [12] containing a promoter and terminator for transcription of T7 RNA polymerase, a translation enhancer of T7 phage 10 gene, a β-lactamase gene, and an NdeI / SalI DNA fragment containing adapted to these sites of the uridine phosphorylase gene sequence of Escherichia coli;
- containing:
as a genetic marker, the β-lactamase gene that determines the resistance of E. coli cells transformed with the plasmid pERURPHOl to penicillin antibiotics;
unique recognition sites of restriction endonucleases located at the following distance to the right of the NdeI site: XbaI - 38 bp , BglII - 96 bp RvuII - 741 bp , PstI - 2288 bp RvuI - 2413 p.p. SalI - 4389 bp

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

 The design of the recombinant plasmid DNA pERURPHOl provides a high level of expression of the uridine 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 of uridine 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) / pERURPHO1 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 haze.

Physico-biological signs. Cells grow at a temperature of from 4 to 40 ^o With optimum pH from 6.8 to 7.5. As a nitrogen source, 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) / pERURPHO1 differs from the recipient strain E. coli BL21 (DE3) only in the presence of recombinant plasmid DNA pERUPHO1, 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) / pERURPHO1 are superproducers of uridine phosphorylase. With the induction of isopropylthio-β-D-galactoside, as well as without induction, the effective biosynthesis of uridine 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-7889 from 01/11/2000

 The invention is as follows. Recombinant plasmid DNA pERURPHO1 was constructed, for which the uridine phosphorylase gene was isolated from E. coli chromosomal DNA using PCR with synthetic olitonucleotide primers (see drawing) containing NdeI restriction enzyme sites (N-terminus of the gene, primer A2) and SalI (C- the end of the gene, primer B2), the DNA obtained is cleaved with the corresponding restriction enzymes and then ligated with the pET-20b (+) vector plasmid cleaved at the same sites [12].

The competent E. coli BL21 (DE3) cells are transformed with a ligase mixture and plated on YT agar containing 50 μg / ml ampicillin or another penicillin antibiotic. The obtained clones are analyzed by hybridization with ³² P-labeled oligonucleotides A2 and B2 (see drawing), and plasmid DNA is isolated from hybridizing clones, which is subjected to restriction analysis using restriction enzymes and BglII. The producer strain E. coli BL21 (DE3) / pERURPHOl is grown in a rich medium (YT-, LB-broth, etc.) (or isopropylthio-β-D-galactoside is induced and re-grown) to achieve maximum culture density.

Isolation of uridine 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 uridine 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 uridine 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 pERURPHO1.

 The chemical synthesis of oligonucleotides is performed by the solid-state phosphoamidite method on an ASM-102U DNA synthesizer (BIOSET, Novosibirsk) with the oligonucleotide chain being extended from the 3'-end to the 5'-end using protected phosphamidites - 5'-dimethoxytrityl-N-acyl-2 '-deoxynucleoside-3'-O- (β-cyanoethyl diisopropylamino) - tetrazole activated phosphites. The synthesis is carried out on a scale of 0.5-0.7 μmol, using porous glass (pore size 500 Å) as a support, to which the first nucleoside unit (load 20-30 μmol / g) is connected via a 3'-succinate bond. Use the synthetic cycle described in [9].

To prepare the DNA vector of plasmid pET-20b (+) [12] (3 μg, 1 pmol) is treated in 40 μl of buffer R (10 mm Tris-Hcl, pH 8.5, 10 mm MgCl ₂ , 100 mm KCl, 0, 1 mg / ml BSA) with restriction enzyme NdeI, and then in 40 μl of Bufur 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 act.) for 1 h at 37 ^o C. Vector fragment size of 3.7 T. p. 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 a fragment of the uridine phosphorylase gene, PCR amplification is performed using E. coli chromosomal DNA (0.01 μg in the sample) as a template, and synthetic oligonucleotides A2 and B2 (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 s at 60 ^° C, elongation - 40 s 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 uridine 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. Act. T4-DNA ligase for 12 hours at 10 ^o C.

An aliquot of the reaction mixture was used to transform competent E. coli cells BL21 (DE3). Transformants are plated on YT agar plates containing 50 μg / ml ampicillin. Recombinant screening is performed by in situ hybridization of colonies with ³² P-labeled oligonucleotide A2 (see drawing). From hybridizing clones, pERURPHOl plasmid DNA was isolated and analyzed with NdeI and SalI endonucleases.

 Example 2. Obtaining strain E. coli BL21 (DE3) / pERURPHO1 (VKPM B-7889), a producer of uridine phosphorylase and determination of its productivity.

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

The producer strain E. coli BL21 (DE3) / pERURPHOl 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 dye bromphenol blue are treated with ultrasound for 20 minutes, heated for 3 min at 100 ^° C and 1 μl samples are used for electrophoresis in 15% SPS-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 uridine 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 s 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. Kremlitsky T. A., Koszatska G. W., Tuttle J. V., Rideout J. L., Eliot G. B. // Carbohydrate Res. , 1981, v. 97, p. 139-146.

 3. Walton L., Richards C. A., Elwell L. P. // Nucl. Acids Res. , 1989, v. l7, p. 6741.

 4. Hutchinson D. W. // Trends Biotechnol. , 1990, v. 8, p. 348-353.

 5. Mikhailopulo I. A., Zinchenko A. I., Kozimierszuk Z., Barai V. N., Bokut S. B., Kalinichenko E. N. // Nucleosides a. Nucleotides, 1993, v. 12, p. 417-422.

 6. Jap. Pat. 5170767, July 9, 93.

 7. Jap. Pat. 06217784, 08/09/94.

 8. US Pat. 4374315, 08.31.82.

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

 10. Veiko V.P., Chebotaev D.V., Ovcharova N.V., Gulko L.B. // Bioorgan. Chemistry, 1998, v. 24, p. 381-387.

 11. Vita A., Amici A., Cacciammani T., Lanciomtti M., Magni G. // Int J. Biochem. 1986, v. 18, p. 431-435.

 12. Novagen Catalog 1996-1997.

Claims (3)

 1. A method of producing E. coli uridine phosphorylase, comprising culturing in a rich medium a producer strain obtained by transformation of Escherichia coli cells with plasmid DNA followed by cell destruction in a buffer solution using ultrasound, characterized in that a specially designed DNA is used as plasmid DNA PERURPHO1, the strain Escherichia coli BL 21 (DE3) is used as the producer strain, and the soluble fraction is separated.  2. Recombinant plasmid DNA pERURPHO1 encoding the amino acid sequence of E. coli uridine phosphorylase, having a molecular weight of 2.94 MDa, consisting of an NdeI / SalI DNA fragment of plasmid pET20b (+), containing the T7 RNA polymerase promoter and transcription terminator, phage T7 gene 10 translation enhancer, β-lactamase gene, and NdeI / SalI DNA fragment containing the uridine phosphorylase gene sequence of Escherichia coli adapted to these sites; as a genetic marker, the β-lactamase gene that determines the resistance of E. coli cells transformed with plasmid pERURPHO1 to penicillin antibiotics; unique recognition sites of restriction endonucleases located at the following distance to the right of the NdeI site: Xbal - 38 bp , BglII - 96 bp , PνuII - 741 bp , PstI - 2288 bp . , PνuI - 2413 bp SalI - 4389 bp  3. The strain Escherichia coli BL21 (DE3), containing recombinant plasmid DNA pERURPHO1, a superproducer of uridine phosphorylase (VKPM B-7889).