RU1387414C - Recombinant plasmid dna psth 2191 encoding synthesis of human somatotropin, and the strain of escherichia coli - a producer of human somatotropin - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: recombinant plasmid DNA pSTH 2191 and strain E. coli containing this plasmid are constructed. Plasmid is modified by such manner that the sequence of site located between trp-promoter and initiating ATG-codon incorporated before the sequence encoding human somatotropin is changed with the sequence that provides more high synthesis of the end product. Strain-producer of human somatotropin is prepared by transformation of E. coli K 802 cells with plasmid pSTH 2191. The level of hormone synthesis at density 4 x 10⁸ cells/ml is 15-20 mg/l. Strain is deposited in All-Union Collection of microorganisms at number BKM CR-267D. EFFECT: increased yield of the end product. 3 dwg

Description

 The invention relates to the microbiological and medical industries, genetic engineering and biotechnology, in particular to recombinant plasmid DNA encoding a variant of human growth hormone, a strain of Escherichia coli containing this plasmid, a producer of human growth hormone.

 The purpose of the invention is to increase the yield of the target product.

 The increase in the growth hormone yield is ensured by modifying the pSTH 19-I / P-trp DNA plasmid so that the sequence of the region located between the trp promoter and the initiating ATG codon inserted before the sequence encoding the human growth hormone (translation initiation region) is replaced by another nucleotide a sequence that provides higher synthesis of the target product.

The essence of recombinant plasmid DNA pSTH 2191 (see Fig. 1) is that it contains a replicon of plasmid pBR 322, a fragment containing the promoter of the tryptophan operon of E. coli and a fragment of human somatotropin DNA encoding from the 2nd to the 191st the amino acid of the hormone in front of which the ATG codon is embedded (see figure 3), namely consists of the following elements:
plasmid DNA of vector pBR 322 from 29th to 4168 bp, the size of which is 4140 bp,
of a DNA fragment containing the E. coli trp promoter inserted between the bla gene of plasmid DNA pRB 322 and a single 1176 bp EcoRI cleavage site; EcoRI - Hind III - a fragment containing DNA encoding from 2 to 191 the amino acid STG, 713 bp in size, in front of which the CAATTCTATG sequence is inserted.

 The size of the hybrid plasmid pSTH 2191 is about 6000 bp

 The copy number of the plasmid is about 20 molecules per cell.

 The plasmid pSTH 2191 DNA contains unique restriction sites EcoRI, Hind III, BamHI, EcoRV, BglII, HpaI, SalGI and SmaI and 2 restriction sites Pst I. The positions of all of the listed restriction sites relative to the EcoRI site are shown in Fig. 1.

 The composition of plasmid pSTH 2191 includes the following genes: bla-gene, which provides the synthesis of β-lactamase (resistance to ampicillin); tet gene providing resistance to tetracycline; STH-modified human growth hormone gene encoding met-STG2-191.

The strain - the producer of human growth hormone - was obtained by transformation of E. coli K 802 cells with plasmid pSTH 2191. The level of hormone synthesis according to simple radial immunodiffusion in the producer strain is 15-20 mg / l at a culture density of 4x10 ⁸ cells / ml. The strain is deposited in the All-Union collection of microorganisms at the IBPM Academy of Sciences of the USSR under the number VKM CR-267D.

 PRI me R 1. Construction of recombinant plasmid DNA pSTH 2191.

 The design scheme is shown in FIG. 2.

10 μm of the plasmid pSTH 19-1. P-trp is digested with EcoRI restriction enzyme in 50 μl of the incubation mixture of the following composition: 100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 1 mM dithiothreitol, 10 units. restriction enzymes EcoRI, for 1 h at 37 ^about C. The completeness of hydrolysis is checked by electrophoresis in 1% agarose gel.

2.5 μl of a 0.25 M EDTA solution, 50 μl of a phenol-chloroform mixture (1: 1) are added to the mixture, shaken for 30 s and the phases are separated by centrifugation. The aqueous phase is extracted with an equal volume (50 μl) of chloroform, then 5 μl of 3M sodium acetate is added to it. pH 6.0, 150 μl of ethanol. The mixture was cooled at - 70 ^C for 10 min and the DNA was precipitated by centrifugation in a centrifuge "Eppendorf" for 10 minutes.

DNA was dissolved in 50 μl of bovine serum albumin solution (500 μg / ml), 50 μl of buffer containing 24 mM CaCl ₂ , 24 mM MgCl ₂ , 0.4 M NaCl, 40 mM Tris-HCl, pH 8.0 and 2 was added mM EDTA, and the mixture was incubated at ²⁰ ° C for 3 min. Then add 1 μl of the preparation of nuclease BaI 31 (100 units / ml). After 1, 2, 3, 4, and 5 min, 20 μl of the incubation mixture were taken and transferred to tubes with 2 μl of 0.2 M EGTA, pH 8.0, cooled in ice.

 The incubation mixtures are combined and the DNA is purified by extraction with an equal volume of a phenol-chloroform mixture (1: 1), chloroform and reprecipitated with alcohol.

DNA is dissolved in 50 μl of water. To 10 ml of a DNA solution add 2 μl of a buffer containing 500 mM NaCl, 100 mM Tris-HCl, pH 7.5, 100 mM MgCl ₂ and 10 mM dithiothreitol, 1 μl of 1 mM dATP, dCTP, dTTP and dGTP solutions, 2 μl of H ₂ O and 2 μl (1 unit / μl) of the Maple fragment of E. coli DNA polymerase 1.

The mixture was incubated at room temperature for 30 minutes. Then, 10 μl of a solution of forced-synthesized 5-ATCGAATCGAT-3 synthetic oligonucleotide (1.0 μg) in 66 mM Tris-HCl buffer, pH 7.5, containing 1 mM ATP, 10 mM MgCl ₂ and 15 mM dithiothreitol, 2 μl 10 mM ATP solution and 2 μl of T4 phage DNA ligase (1 unit / μl). The mixture was incubated at room temperature for 6 hours, then 1 μl of a 0.25 M EDTA solution was added to it and treated with an equal volume of phenol-chloroform mixture and an equal volume of chloroform.

DNA is precipitated with ethanol as described previously and dissolved in 20 ml of H ₂ O.

 Next, the DNA is hydrolyzed with restriction enzymes CoRI (30 units) and EcoRV (2 units) under standard conditions, the mixture is treated with phenol-chloroform, chloroform and reprecipitated with ethanol.

DNA is dissolved in 10 μl of H ₂ O and fractionated by electrophoresis in a 5% polyacrylamide gel, the gel is stained with ethidium bromide, a gel strip containing 5100-5200 bp fragments is cut out and eluted from the gel. Thus, a mixture of plasmids for cloning a fragment encoding somatotropin is obtained.

 In the same polyacrylamide gel, the double EcoRI-EcoRV hydrolyzate of the starting plasmid pSTH 19-I / P-trp (2 μg) was separated and a smaller fragment of 869 bp containing the nucleotide sequence encoding human somatotropin was isolated from the gel.

After reprecipitation with alcohol, DNA fragments are dissolved in 10 μl of H ₂ O. Ligation of DNA is carried out in an incubation mixture of the following composition: 1 μl of a plasmid solution (5100-5200 bp; v.), 1 μl of a solution of EcoRI-EcoRV fragment (860 bp ), 2 μl of 10 x ligase buffer, 15 μl of H ₂ O, 1 μl of T4 phage ligase DNA (1 unit / μl); 10 x ligase buffer contained 0.66 mM Tris-HCl, pH 7.5, 50 mM MgCl ₂ , 50 mM dithiothreitol and 10 mM ATP. Incubate 4 hours at 14 ^{° C} and then for 12 hours at 4 ° C. To ^the incubation mixture was added 30 mg of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and transformed cells E.Coli HB 101 strain.

50 ml YT-medium inoculated with 1-2 ml of an overnight culture of the strain E.coli HB 101 cells and grown on a shaker at ³⁷ ° C to a density of 0.6 at 550 nm. Cells are harvested by centrifugation in the cold at 10,000 rpm for 10 minutes. The precipitate was suspended in 25 ml of a cold solution: 50 mM Tris-HCl, pH 7.5, 50-70 mM calcium chloride, and kept in ice for 30 minutes. Subsequent operations are also carried out in an ice bath. Then the cells are again precipitated in the same mode and suspended in 2.5 ml of the same solution and left for 30 minutes. The ligation mixture was in a volume of 50 l was added to 100 l of the resulting cell suspension and incubated for 20 minutes on ice, followed by 2 minutes at 42 ^{° C,} then 10 minutes at room temperature. To each sample was added 2 ml of fresh YT medium, and cells were grown for 1 hour shaking at 37 ° ^C. Cells from the source tubes and the tubes of the dilution of 1:10 and 1: 100, plated on YT agar medium (5 g of yeast extract , 8 g of bactotriptone, 5 g of NaCl, 15 g of bactoagar per 1 liter of medium) and antibiotics (20 mg of ampicillin and 20 mg of tetracycline per 2 l), 0.5 ml per cup. Colonies grown on the plates after 1420 hours at 37 ° ^C.

Ampicillin and tetracycline resistant columns are selected and the hormone content analyzed. For this, cells are grown in 5 ml of medium (0.5% yeast extract, 0.8% bactotryptone, 0.5% sodium chloride, pH 7.6) containing 20 mg / l ampicillin and 7 mg / l tetracycline, at 37 ^about C to an optical density of 1 at 550 nm.

Cells are pelleted by centrifugation at 10,000 rpm for 10 minutes. The cell pellet was suspended in 0.2 ml of solution 1 (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0). Then add 0.2 ml of solution 2 (0.2 N. NaOH, 0.02% SDS) and the suspension is incubated for 5 min at 0-5 ^about C. Then add 0.1 ml of solution 3 (150 mm Tris-HCl, pH 8.0, 280 mM MgCl ₂ 4 mM CaCl ₂ ). After 5 min, the suspension was centrifuged for 10 min at 12000 × G, the supernatant was collected and transferred to a clean tube.

To prepare the agar plate, two glasses measuring 6x9 cm or 9x12 cm with 1.5 mm thick gaskets laid at the edges are fastened with metal clips and sealed with adhesive or tape on the bottom. Prepare a 1.5% solution of bacto agar (Difco) in 10 mM Na-phosphate buffer, pH 7.2, it was cooled ^to 40 ° C, added 1/20 hours. The aqueous solution was lyophilized rabbit serum for growth hormone (40 mg / ml ), the resulting mixture is poured into the mold. After hardening of the agar, one of the glasses is removed and cylindrical wells 1.8 mm in diameter at a distance of 1 cm from each other are cut out in the thickness of the agar. In wells N 1-5 contribute 6 μl of standard solutions of STH with concentrations of 200, 100, 50, 25 and 12.5 μg / ml, respectively. 6 μl of cell lysate was added to well No. 6.

The plate was incubated in a moist chamber at 37 ^C. After 4 hours the agar formed visible to the naked eye circles precipitation. The diameter of the precipitation circles is measured, and according to the data for standard STH solutions, a calibration curve is constructed of the square of the diameter of the circle on the concentration of the hormone, which determines the concentration of the hormone in the lysate.

 Cells from 3 ml of overnight culture are pelleted in an Eppendorf benchtop centrifuge at 10,000 x 2 minutes. The cell pellet is suspended in 100 μl of a 1: 2 mg / ml lysozyme solution, 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0. Test tubes are placed on ice for 30 minutes. Subsequent operations are also carried out in ice. Then add 200 μl of a solution of 2: 0.2 N. sodium hydroxide, 1% sodium dodecyl sulfate, and mix well. After 5 min, add 150 μl of a 3M solution of sodium acetate, pH 4.8, mix and leave for 1 h. Then the solution is centrifuged for 5 min at 10000xG. From the supernatant, the plasmid is precipitated with alcohol and dissolved in 50 μl of water. Thus, about 50 μl of the plasmid was isolated. Next, the isolated plasmids were analyzed by electrophoresis in a 1% agarose gel without preliminary digestion and after digestion with restriction enzymes.

 To clarify the structure of plasmid DNA, the nucleotide sequence is analyzed in the region between the EcoRI and HpaI sites.

Based on the determination of the hormone content, restriction analysis, and determination of the primary structure, a plasmid designated pSTH 2191 was selected that encodes the synthesis of growth hormone, which is a mixture of two variants of the hormone (des-phen ¹ ) somatotropin and (des-phen ^1- pro ² ) somatotropin approximately equally.

 PRI me R 2. Obtaining a strain of E. coli K 802 - producer of human growth hormone that does not contain N-terminal methionine.

 Plasmid pSTH 2191 is transformed into E. coli K 802 strain according to the method described in example 1, and a human somatotropin producing strain is obtained. The strain is deposited in the All-Union collection of microorganisms at the IBPM Academy of Sciences of the USSR under the number VKM CR-267D.

The hormone content in the biomass is determined by lysis of bacteria and measurement of the hormone in the lysate by simple radial diffusion in agar, as described in example 1. The hormone content is 15-20 mg per 1 liter of culture when the strain is grown to a density of OD ₅₅₀ = 1, which 2.2 times the hormone content in the strain of the prototype VKM R 26-D.

 The E. coli K 802 strain containing the recombinant plasmid pSTH 2191 is characterized by the following features.

 Morphological signs of the cell are straight, rod-shaped, gram-negative, non-spore-bearing.

 Cultural signs. Cells grow well on commonly used culture media. When growing on the Difco nutrient agar, the colonies are smooth, round, shiny, gray, the edges of the colonies are even. When growing in liquid media, UT and LB form a uniform intense haze.

Physico-biochemical characteristics. The optimal cultivation temperature is 37 ^{° C,} the optimum pH of from 6.8 to 7.5.

 As a carbon source, many carbohydrates, alcohols, and organic acids are used.

 As a source of nitrogen, both mineral salts in ammonium or nitrate form, and organic salts in the form of peptone, tryptone, amino acids, are used.

 Antibiotic resistance. Resistant to ampicillin due to the presence of a plasmid. Resistant to tetracycline 20 mg / l on agar, 5-7 mg / l in a liquid medium.

 To obtain human growth hormone that does not contain N-terminal methionine, the VKM CR 267D strain is cultured on a nutrient medium of known composition, the bacterial biomass is collected by centrifugation, the cells are destroyed and proteins are extracted from them, and the hormone is purified from bacterial proteins using known methods.

Claims (2)

 1. Recombinant plasmid DNA pSTH 2191, encoding the synthesis of human growth hormone, size 6.0 TPO, consisting of: plasmid DNA vector pBR 322 size 4.14 TPO .; a 1.17 kbp DNA fragment containing E. coli trp promoter, EcoRI-Hind III fragment of 713 bp human reconstructed human somatotropin DNA, 1 EcoRI restriction enzyme digestion site; 1 plot cleavage of the restriction enzyme Hind III, located at a distance of 713 bp clockwise from the EcoRI site, 1 BamHI restriction enzyme cleavage site located at a distance of 1059 bp clockwise from the EcoRI site, 1 SalGl restriction enzyme cleavage site, located at 1334 bp clockwise from the EcoRI site, 2 PstI restriction enzyme cleavage sites located at 139 and 4302 bp clockwise from the EcoRI site, 1 HpaI restriction enzyme cleavage site located at 39 bp counterclockwise from the EcoRI site and having genetic markers: bla gene for the synthesis of β-lactamase (resistance to ampicillin), located in the area from 1200 to 2100 bp to the left of the EcoRI site, the tet gene that provides tetracycline resistance, located on a plot of about 1000 bp to the right of the Hind III site; In front of the somatotropic hormone DNA sequence, the promoter and operator regions of the E. coli trp operon are located to the left of the EcoRI site; the region between the trp promoter and the initiating codon has the following nucleotide sequence: + I AGTACGCAAGTTCACGTAAAAAGGGTATCGAATTCT.  2. The strain Escherichia coli VKM CR-267D - producer of human growth hormone.

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