RU2200196C2 - Recombinant plasmid dna ppho-sod 23 encoding synthesis of human superoxide dismutase and strain saccharomyces cerevisiae = d-11 vniischm as producer of human superoxide dismutase - Google Patents

Abstract

FIELD: molecular biology, biotechnology, biochemistry, microbiological and medicinal industry. SUBSTANCE: invention relates to technology of preparing superoxide dismutase preparations (SOD). Recombinant plasmid DNA pPHO-SOD 23 encoding synthesis of human superoxide dismutase consisting of Eco RI/Bam HI-fragment p33 of expression cassette with gene SOD, Hind III/Bam HI-fragment with sequence of alcoholdehydrogenase transcription terminator and Bam HI-fragment with sequence of hepatitis B Hbs- antigen. The strain of yeast Saccharomyces cerevisiae = D-11 VNIISCHM as a producer of superoxide dismutase obtained by transformation of cells of recipient strain 20B12 Saccharomyces cerevisiae with genotype trPU with plasmid DNA pPHO-SOD23 exhibits the ability to selective maintaining expression plasmid in media of any composition no containing tryptophan. The strain allows to enhance the yield of SOD significantly (SOD content in lysate is 4 200 mcg/ml of lysate). EFFECT: enhanced yield of enzyme. 2 cl, 2 dwg, 1 ex

Description

 The invention relates to the microbiological and medical industries, in particular to a technology for producing superoxide dismutase (SOD) preparations.

 The currently described yeast strain producing human superoxide dismutase synthesizes the target product constitutively, which limits growth, reduces biomass yield and, accordingly, reduces productivity and genetic stability.

 It is known to obtain SOD based on yeast strains (Biotechnology, 1987, v.5, p.363-366).

 The disadvantage of this technology is the need to use expensive selective media, as well as the relatively low stability of the strain.

 The prototype of the claimed group of inventions is a strain of Saccharomyces cerevisiae YBS13Chgk / pBr-SOD-PGKdel and the expression plasmid pBr-SOD-PGKdel used in its creation (US Pat. RF 2044771, 1995, class C 12 N 1/16).

 The disadvantage of this strain and used in this plasmid is the lack of stability of properties during cultivation.

 The problem solved by the authors was the creation of a plasmid and strain, allowing to obtain SOD with a fairly high yield on relatively cheap media with a higher stability of properties during cultivation.

 The problem was solved by creating a yeast expression plasmid pPHO-SOD23 containing the human superoxide dismutase gene sequence controlled by the yeast phosphatase promoter and the alcohol dehydrogenase transcription terminator, and also containing the hepatitis B Hbs antigen sequence to stabilize the plasmid construct in yeast cells; transformation of the yeast strain Saccharomyces cerevisiae 20B12 with this plasmid; and, as a result, the creation of a yeast strain of a producer of human superoxide dismutase.

The scheme of the plasmid pPHO-SOD23 is shown in figure 1, where a - corresponds to the EcoRI / BamHI fragment of the commercial plasmid p33 and contains:
A1 - yeast phosphatase promoter pRNO5,
A2 is a fragment of a yeast 2 μm B-type plasmid containing ori of this plasmid,
A3 - yeast active gene TRP1,
A4 is a fragment of plasmid pBR322 containing ori and the BLA gene of this plasmid,
A5 — yeast phosphatase transcriptional terminator of tPHO5;
B - corresponds to the sequence of Hbs-antigen of hepatitis B, flanked by BamHI sites;
C - corresponds to the sequence of the alcohol dehydrogenase transcription terminator (Hind ^III / BamHI fragment);
D is an expression cassette with the SOD gene, flanked by EcoRI / KpnI sites, which has the following form (see sequence 1).

 Plasmid pPHO-SOD23 was obtained as follows. Complementary DNA (cDNA) was synthesized by reverse transcription on a messenger RNA matrix isolated from human placenta. The SOD gene was obtained by polymerase chain reaction (PCR) on a human placenta cDNA matrix with seeds representing the start and end sequences of the human SOD gene containing its initiation site (ATG) and translation termination (TAATGA). HindIII adapters were sewn onto the product obtained by PCR and the human SOD gene was cloned into the HindIII site of the commercial plasmid pAAN5. Then, an EcoRI-BamHI fragment of the obtained plasmid containing the SOD gene and yeast alcohol dehydrogenase transcription terminator was combined with a large EcoRI-BamHI fragment of the commercial p33 plasmid. A BamHI fragment of plasmid p33 containing the sequence of hepatitis B Hbs antigen was inserted into the obtained plasmid.

 The yeast strain Saccharomyces cerevisiae 20B12 of the trp1 genotype was used as the recipient strain during transformation with plasmid pPHO-SOD23. Transformants were selected for growth ability on tryptophan-free media. One of these clones was selected as a producer and designated 20B12 / pPHO-SOD23.

Based on the construction method, in the case of loss of the plasmid, a recipient cell of the trp ^- genotype ^{is formed} ; such cells are not capable of biosynthesis of tryptophan and are not viable in selective medium. Thus, the created producer strain has the ability to selectively maintain the expression plasmid on media of any composition not containing tryptophan.

 The strain Saccharomyces cerevisiae 20B12 / pPHO-SOD23 was deposited in the collection of the All-Russian Research Institute of Agricultural Microbiology (VNIISKhM) under the number D-11 VNIISKHM.

 The strain is characterized by the following cultural-morphological and physiological-biochemical characteristics.

 Vegetative cells of a two-day culture grown on solid nutrient medium with 2% glucose as the sole carbon source have an oval shape, cell size 10-15 microns, protoplasm homogeneous, reproduction by budding.

When grown on a rich solid YEPD medium at 30 ^{° C.} after 72 hours of growth, the colonies have the following appearance: white colonies with a smooth or slightly serrated edge, a dull surface, raised, opaque.

Growth in a liquid selective glucose-mineral medium without phosphate containing 0.1% yeast extract at 28-32 ^o C: during the first 24 hours of cultivation, the liquid is cloudy, the precipitate is white, does not clump, does not form wall films. The growth temperature of 25-33 ^o C (optimum 28 ^o C), the pH of the cultivation of 4.8-7.0 (optimum 6.0).

 Assimilation of carbon sources: ferments glucose, galactose, fructose, maltose, sucrose, dextrins, starch, assimilates ethanol, glycerin, lactate; Does not assimilate acetate.

 Assimilation of nitrogen sources: assimilates amino acids, urea, ammonium salts; nitrates does not restore.

The strain Saccharomyces cerevisiae D-11 ARRIAH is not pathogenic. The strain is stored on agar rich YEPD medium with glucose for 3 months at + 4 ^o C.

 The possibility of practical use of the strain is illustrated by the following example.

Example
The cells of the producer strain D-11 VNIISKhM and the initial strain 20B12 were grown in flasks at 29 ± 1 ^° C and aeration on 1 liter medium of the following composition: calcium dihydrogen phosphate 1 g, sodium chloride 0.1 g, ammonium sulfate 5 g, casamic acids (or peptone - HCl hydrolyzate) 10 g, anhydrous glucose 20 g, magnesium sulfate 0.5 g, calcium chloride 0.1 g, potassium iodide 100 μg, boric acid 10 μg, iron sulfate 50 μg, manganese sulfate 10 μg, uracil 20 mcg, inosine 10 mcg, kanamycin 50 mcg, riboflavin 0.2 mcg, nicotinic acid 0.2 mcg, 4-aminobenzoic acid 0.2 mcg, panteto atm calcium 0.2 mg, 0.2 mg pyridoxine, thiamine bromide 0.2 ug, biotin 0.002 micrograms. The initial titer of the cultures was 10 ² cells / ml. After 18-20 hours of cultivation (5 × 10 ⁶ cells / ml), the cultures were transferred to a medium containing 1 l: potassium chloride 1 g, sodium chloride 0.1 g, ammonium sulfate 5 g, casamic acids (or peptone – HCl hydrolyzate) 10 g, yeast extract 1 g, zinc sulfate 16 mg, anhydrous glucose 20 g, magnesium sulfate 0.5 g, calcium chloride 0.1 g, potassium iodide 100 μg, boric acid 10 μg, iron sulfate 50 μg, sulfate manganese 10 mcg, uracil 20 mcg, inosine 10 mcg, kanamycin 50 mcg, riboflavin 0.2 mcg, nicotinic acid 0.2 mcg, 4-aminobenzoic acid 0.2 mcg, calcium pantetonate 0.2 mcg, pyridoxine 0.2 mcg, thiamine bromide 0.2 mcg, biotin 0.002 mcg. In this case, the initial culture was bred 5 times, to a titer of 1 • 10 ⁶ cells. / ml After 20-24 hours of cultivation, the cultures transferred to the stationary phase of growth at a titer of 1.2 • 10 ⁸ cells / ml. The yield of yeast biomass, 80% moisture, was 15 g / l. The biomass collected during this cultivation period was used for superoxide dismutase analysis. Cells from 20 ml of culture were collected by centrifugation, washed with water, suspended in 20 ml of 20 mM phosphate buffer and destroyed after adding an equal volume of glass beads on a Vortex vibrator 4 times for 5 minutes. To obtain a soluble protein fraction, debris was separated by centrifugation at 16,000 rpm for 20 minutes. The cell extract thus clarified was analyzed electrophoretically and the content of superoxide dismutase was determined in it. According to the results of electrophoresis (figure 2, where lane 1 is a pure preparation of human SOD, lane 2 is an extract of cells of strain 20B12, lane 3 is an extract of cells of strain D-11 VNIISCHM) it can be seen that the producer strain contains a significant amount of human superoxide dismutase, which is absent in extract of the original strain. From the densitometry data of strip 3 of this electrophoregram, it follows that human superoxide dismutase makes up 20% of soluble cellular protein.

 The presence of superoxide dismutase in cell extracts was measured using an enzyme-linked immunosorbent assay for the interaction of the target protein with a pair of monoclonal antibodies, one of which is conjugated to horseradish peroxidase, which allows quantitative testing of the presence of human superoxide dismutase in samples. It was found that the content of SOD in the lysate is 4200 μg per ml of lysate.

Claims (2)

 1. Recombinant plasmid DNA pPHO-SOD23, encoding the synthesis of human superoxide dismutase, consisting of EcoR I / BamH I fragment of the commercial plasmid pZZ, expression cassette with the SOD gene (EcoR I / Kpn fragment I), the sequence of the alcohol dehydrogenase transcription terminator (Hind III / Bam fragment) and the sequence of Hbs hepatitis B antigen (BamH I fragment).  2. The yeast strain Saccharomyces cerevisiae D-11 VNIISCHM - producer of superoxide dismutase.

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