A method of obtaining a recombinant polynucleotide-phosphorylase, recombinant plasmid dna perpupho1 and the escherichia coli strain bl21(de3)/perpupho1 for its implementation

 

(57) Abstract:

The invention relates to biotechnology, in particular genetic and protein engineering, and solves the problem of obtaining highly productive recombinant bacterial strain-producer of polynucleotide-phosphorylase. Recombinant plasmid DNA ERPUPHO1 encoding the amino acid sequence of polynucleotide-phosphorylase E. coli, consists of: NcoI/EcoRI-fragment DNA plasmids 23d containing the promoter and terminator of transcription of the T7-RNA polymerase, power broadcast of gene 10 of phage T7 gene-lactamase, and NcoI/EcoRI fragment containing DNA adapted to these sites the sequence of the gene polynucleotide-phosphorylase of Escherichia coli. Producing strains of E. coli BL21(DE3)/ERPUPHO1 obtained by transformation of cells of E. coli plasmid DNA ERPUPHO1, cultivated prior to the accumulation of recombinant polynucleotide-phosphorylase in the amount of 60-70% of the total protein, the cells are destroying ultrasound into the buffer solution and separating the soluble fraction. Producing strains of E. coli BL21(DE3)/ERPUPHO1 grown in rich medium (YT-, LB-broth and others ) (or induce isopropylthio--D-galactoside, and again is grown) to achieve maximum culture density. The invention allows to obtain purin the tion relates to biotechnology, in particular, genetic and protein engineering. It includes a constructed in vitro recombinant plasmid DNA pERPUPHO1 contributing to the biosynthesis of polynucleotide-phosphorylase a strain of E. coli, the E. coli strain BL21(DE3)/pERPUPHO1 - superproducer polynucleotide-phosphorylase and the method of obtaining polynucleotide-phosphorylase on the basis of the above recombinant DNA and producer strain for the reaction of transglycosylase in the synthesis of nucleosides.

Polynucleotide-phosphorylase Escherichia coli (EC 2.4.2.1) is a protein with a mol. m 24 KDa, functioning as hexamer mol. m 122 KDa [1] and catalyzes the catabolic reaction postrelease purine nucleosides in the cells of E. coli [1,2] . The enzymatic activity of polynucleotide-phosphorylase allows its use in the reaction of transglycosylase in the synthesis of modified nucleosides, which are used in medicine as therapeutic drugs [3] .

For practical purposes, until recently, was used not only isolated from the E. coli polynucleotide-phosphorylase, but mainly the enzymatic activity of whole bacterial cells of E. coli, or a mixture of nucleoside-phosphorylase of these cells without enrichment or separation [5-12].

The present invention solves the problem of obtaining highly productive recombinant bacterial strain-producer, allowing to obtain recombinant polynucleotide-phosphorylase with high yield and simplified technology.

The problem is solved due to the fact that producing strains obtained by transformation of Escherichia coli cells plasmid DNA, cultivated prior to the accumulation of recombinant polynucleotide-phosphorylase in the amount of 60-70% of the total protein of cells destroy the cells in a buffer solution and separating the soluble fraction. The content in this fraction polynucleotide-phosphorylase is up to 80% of the total protein content, and enzyme can be used without further purification or purified to a homogeneous state by standard methods.

Use producing strains of Escherichia coli BL21(DE3) containing plasmid DNA pERPUPHO1 - superproducer polynucleotide-phosphorylase E. coli.

Closed-phosphorylase E. coli

- having a molecular weight 2,90 MDA;

- consisting of:

NcI/EcoRI-fragment DNA plasmids pET23d(+)[14] , containing the promoter and terminator of transcription of the T7-RNA polymerase, power broadcast of gene 10 of phage T7 gene-lactamase, NcoI/EcoRI fragment containing DNA adapted to these sites the sequence of the gene polynucleotide-phosphorylase of Escherichia coli,

- contains:

as a genetic marker gene-lactamase determining the stability of the transformed plasmid to the cells of E. coli to penicillin antibiotics;

unique recognition sites of restriction endonucleases that are located in the following distance to the right of the NcoI site: XbaI - 38 p. O. , BglII - 96 p. O. , PvuII - 741 p. O. , BglI - 2163 p. O. , PvuI - 2413 p. O. , EcoRl - 4389 p. O.

Use producing strains of Escherichia coli BL21 (DE3) containing the recombinant plasmid DNA pERPUPHO1 producing polynucleotide-phosphorylase.

The invention allows to obtain recombinant polynucleotide-phosphorylase on simple technology and high output.

Construction of recombinant plasmid DNA pERPUPHO1 provides a high level of expression of cloned it gene polynucleotide-phosphorylase.

To construct plasmids the optimum regulatory elements, controlling his expression.

The source of the structural gene polynucleotide-phosphorylase is chromosomal DNA of E. coli. Recombinant gene allocate by PCR using synthetic oligonucleotide primers and then clone into a vector plasmid pET-23d(+) [14] .

Offer producing strains of Escherichia coli BL21(DE3)/pERPUPHO1 characterized by the following features:

Morphological features. Cells are rod-shaped, gram-negative, risperadone.

Cultural characteristics. Cells grow well on simple nutrient media. During growth on agar "Difco" - colonies are round, smooth, dull, shiny grey, smooth edge. With the growth in liquid medium (minimal medium with glucose or YT-broth) intensive form a smooth suspension.

Physical and biological characteristics. Cells grow at temperatures from 4 to 40oC at the optimum pH of from 6.8 to 7.5. As the source of nitrogen used as a mineral salt in ammonium form, and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. as a source of carbon use amino acids, glycerol, carbohydrates.

Resistance to antibiotics. Cells are resistant to Pimienta E. coli BL21(DE3) only in the presence of recombinant plasmid DNA pERPUPHO1, which gives it resistance to penicillin antibiotics.

Strains-producers obtained by transformation of competent cells of E. coli BL21(DE3) corresponding recombinant plasmid DNA.

Cells of E. coli BL21(DE3)/pERPUPHO1 are superproduction polynucleotide-phosphorylase. When induction of isopropylthio --D-galactoside, and without induction is effective biosynthesis polynucleotide-phosphorylase, which accumulates in the cells in more than 60% of the total protein of bacteria.

Producing strains deposited in Russian national collection of industrial microorganisms, N VKPM B-7888 from 11.01.2000,

The invention is as follows. Construct recombinant plasmid DNA pERPUPHO1, for which the gene polynucleotide-phosphorylase isolated from the chromosomal DNA of E. coli by PCR using synthetic oligonucleotide primers (see drawing) containing sites restricts Ncol(N-end of the gene, primer A1) and SalI(s-end of the gene, primer B1) obtained DNA digested with appropriate restrictase and then are ligated with the cleaved by the same sites of vector plasmid pET-23d(+) [14] .

Ligase mixture of the penicillin antibiotic. The resulting clones analyzed by hybridization32P-labeled oligonucleotides A1 and B1 (see the drawing), and hybridization of clones secrete plasmid DNA, which is subjected to restriction analysis using restricted Ncol and SalI.

Producing strains of E. coli BL21(DE3)/pERPUPHO1 grown in rich medium (YT-, LB-broth and others ) (or induce isopropylthio -- D-galactoside, and again is grown) to achieve maximum culture density.

The selection polynucleotide-phosphorylase from cells of a producer includes the following stages:

the destruction of cultured cells using ultrasound;

- separation of the soluble fraction by centrifugation; the content in this fraction polynucleotide-phosphorylase is up to 80% of the total protein content, and enzyme can be used without additional purification;

from the soluble fraction of the target protein can be purified to a homogeneous state by standard methods.

The drawing shows the structure of the gene polynucleotide - phosphorylase and synthetic primers used for isolation of the gene by PCR and selection of clones by hybridization.

The invention is illustrated in the following examples.

Example 1. To ofanim fostamatinib method on a DNA synthesizer ASM-102U (BIOSSET, Novosibirsk) with increasing oligonucleotide chain in the direction from the 3'end to the 5'-end using secure phosphamidon - 5'- dimethoxytrityl-N-acyl-2'-deoxynucleoside-3'-O -(-Tianeti-diisopropylamino)-phosphites activated by tetrazole. The synthesis is carried out in the scale of 0.5-0.7 µm, using as a carrier of porous glass (pore size 500 A), to which 3'- succinate connection joining the first nucleoside link (load 20-30 µmol/g). Use synthetic cycle, described in [13] .

For preparation of vector DNA plasmids pET-23d(+) (3 μg, 1 pmol) was worked up in 40 μl of Y buffer (33 mm Tris-acetate, pH of 7.9, 10 mm Mg-acetate, 66 mm K-acetate, 1, 0.5 mm DTT, 0.1 mg/ml BSA) with the restriction enzyme Ncol (10 units act. ), and then 40 μl Bushra R (10 mm Tris-HCl, pH 8.5, 10 mm MgCl2, 100 mm KCl, 0.1 mg/ml BSA) with the restriction enzyme EcoRI (10 units act. ) for 1 h at 37oC. Vector fragment between 3.6 T. p. O. after electrophoresis in 1% agarose gel electrophoretic move in the layer of DEAE-paper, then elute 1M NaCI and precipitate DNA from solution by ethanol.

For the preparation of a fragment of the gene polynucleotide-phosphorylase spend amplification by PCR, using as template the chromosomal DNA of E. coli (0,01 µg / sample), and as a pry is aStore, containing each of the four dNTP at a concentration of 0.5 mm and 5 units of the act. Taq-DNA-polymerase, as follows: denaturation 1 min at 94oC, annealing 30 sec at 60oC, elongation of 40 s at 72oC, 30 cycles of PCR. After that the reaction mixture deproteinizing chloroform, evaporated to dryness, the residue was dissolved in 20 μl of water, then break down the same restrictases that were used in the preparation of the vector and produce the target fragment from the agarose gel.

The obtained synthetic fragment from the genome of polynucleotide-phosphorylase in the amount of 2 pmol was added to a solution of 1 μg of the above described vector fragment in 10 μl of buffer (20 mm Tris-HCl, pH 7,56, 10 mm MgCl2, 0,2 mm gatr, 10 mm dithiotreitol) and are ligated with 10 units of the act. T4-DNA-ligase for 12 hours at 10oC.

An aliquot of the reaction mixture used to transform competent cells of E. coli BL21(DE3). Transformants plated on plates with YT-agar containing 50 μg/ml ampicillin. Screening of recombinants carried out by using hybridization of colonies in situ with32P-labeled oligonucleotide A1 (see drawing). From hybridization of clones secrete DNA plasmids pERPUPHO1 and analyzed by endonucleases NcoI and EcoRI.

Example 2. Obtaining strain E. P> Cells of E. coli BL21(DE3) carrying plasmid pERPUPHO1, the structure of which is confirmed by the data analysis (see example 1) are superproduction polynucleotide-phosphorylase.

The producer strain E. coli BL21(DE3)/pERPUPHO1 grown at 37oC in 100 ml of YT-broth (pH 7.0) with 50 mcg/ml ampicillin for 2 h on a rocking chair with speed 190 rpm to turbidity AND550of 0.7-0.8, add isopropylthio --D-galactoside to a concentration of 0.2 mm and continue the process for another 6 hours Every hour take a sample of 2 ml, AND determine550and the amount of culture, corresponding to 1 ml AND5501,0, centrifuged 5 min at 6000 Rev/min the Precipitated cells in 100 ál lyse buffer with dye bromophenol blue handle 20 with ultrasound, heated 3 min at 100oC and the samples, 1 μl used for electrophoresis in 15% SDS-PAG. Gel stain, Kumasi R-250 according to standard methods and scan to determine the relative amount of protein in the band of the target protein.

Example 3. Getting polynucleotide-phosphorylase.

Wet cells (10 g) is suspended in 20 ml of buffer (30 mm Na - phosphate, pH 7.0, 5% glycerol) and destroy ultrasound ultrasonic disintegrator Sonifier 240 (Branson) (3 pulse for 20 seconds And when 2.0 and 0oC). GOK used for the reaction of transglycosylase.

LITERATURE

1. Jensen K. F. , P. Nygaard //Eur. J. Biochem. , 1975, v. 51. p. 253-265.

2. Kremlitsky T. A. , Koszalska G. W. , Tuttle J. V. , J. L. Rideout , G. B. Elion //Carbohydrate Res. , 1981, v. 97, p. 139 - 146.

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

4. Pal , S., Nair V. // Biocatalysis, a. Biotransform. , 1997, v. 15, p. 147-158.

5. Mikhailopulo I. A. , Zinchenko A. I. , Kozimierszuk Z. , Barai V. N. , Bokut S. B. , E. N. Kalinichenko //Nucleosides a. Nucleotides, 1993, v. l2, p. 417-422.

6. Jap. Pat. 5170767, 09.07.93.

7. Jap. Pat. 06217784, 09.08.94.

8. US Pat. 4374315, 31.08.82.

9. Zinchenko A. L, Barai V. N. , Bokut S. B. , Kvasyuk E. L. Mikhailopulo I. A. Appl. Environ. BiotechnoL, 1990, v. 32, p. 658-661.

10. Eroshevsky L. A. , Baray Century. N. , Zinchenko A. I. , Kasuk E. I. , Mihalopoulos I. A. //Antibiot. The honey. Biotechnol. , 1986, T. 31, S. 174-178.

11. WO Pat. 9421118, 29.09.94.

12. WO Pat. 9507718, 23.03.95.

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

14. Novagen Catalog 1996-1997.

1. The method of obtaining polynucleotide-phosphorylase E. coli, including cultivation in rich medium producer strain obtained by transformation of Escherichia coli cells plasmid DNA with subsequent destruction of the cells in buffer solution using ultrasound, characterized in that the quality of plasmid DNA using a specially designed DNA ERPUPHO1, as the nucleoside-phosphorylase.

2. Recombinant plasmid DNA ERPUPHO1 encoding the amino acid sequence of polynucleotide-phosphorylase E. coli with mol. m 2,90 Hmm, consisting of: NcoI/EcoRI-fragment DNA plasmids 23d(+), containing the promoter and terminator of transcription of the T7-RNA polymerase, power broadcast of gene 10 of phage T7 gene-lactamase, and NcoI/EcoRI fragment containing DNA adapted to these sites the sequence of the gene polynucleotide-phosphorylase of Escherichia coli; contains: as a genetic marker gene-lactamase determining the stability of the transformed plasmid ERPUPHO1 of E. coli cells to penicillin antibiotics; unique recognition sites of restriction endonucleases that are located in the following distance to the right of the NcoI site : XbaI - 38 p. O. , BglII - 96 p. O. , PvuII - 741 p. O. , BglI - 2163 p. O. , PvuI - 2413 p. O. , EcoRI - 4389 p. O.

3. The Escherichia coli strain BL21 (DE3) containing the recombinant plasmid DNA ERPUPHO1, superproducer polynucleotide-phosphorylase E. coli (VKPM B-7888).

 

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