RU2009199C1 - Bitter infusion - Google Patents

Abstract

FIELD: liqueur and vodka production. SUBSTANCE: composition contains (kg/1000 dal) : flag root 0.9-1.1, ST. John's wort 2.7-2.9, above ground part of ginseng 0.5-0.7, calendula 1.0-1.2, tansy 2.5-2.7, matricary 3.8-4.0, sugar 20-. 0-20.4, color 2.0-2.2, aqueous-alcohol liquid the rest. EFFECT: improves bitters quality. 1 dwg, 1 tbl

Description

 The invention relates to biotechnology and genetic engineering and is a recombinant plasmid DNA to obtain somatostatin-14 and a strain of the bacterium Escherichia coli - producer of somatostatin-14.

 Describes the construction of expressing plasmid vectors designed for production under the control of promoters of somatostatin-14 hybrids with various carrier proteins [1, 2, 3, 4, 5, 6, 7].

 The disadvantage of these vectors is, in some cases, the instability of the plasmid and the low level of expression of the hybrid protein in E. coli cells, in others - the complexity of the design and methods for achieving the expression of hybrid genes.

 Somatostatin-14 producing strains are known [1, 2, 3, 4, 5, 6, 7], but both the E. coli RRI prototype strain [1] and other analogous strains have a high level of proteolytic activity.

 However, this strain contains mutations that reduce the degradation of abnormal proteins, mutant proteins, and stably maintains amber fragments.

 The aim of the invention is to obtain recombinant plasmid DNA containing the somatostatin-14 gene, and a somatostatin-14 producer strain using the Escherichia coli strain with a reduced level of degradation of abnormal proteins.

 Designed recombinant plasmid pCCS size 4920 p. N. contains a fragment of the vector pBR 325 with a size of 4860 bp with the β-lactamase gene and a part of the modified chloramphenicolacetyltransferase (CAT) gene, to the 3'-end of which, through the synthetic linker containing the EcoRI restriction site and flanked from the 5'-end of the GG nucleotide sequence, the synthetic somatostatin-14 gene was tuned at the EcoRI site. This plasmid determines the constitutive synthesis of the CAT-somatostatin-14 fusion protein under the control of the CAT promoter in E. coli MKD 3207 cells with a reduced level of degradation of abnormal proteins.

 PRI me R 1. Assembly and molecular cloning of the somatostatin gene.

 Oligonucleotides 1-10 are synthesized by the block triester method in solution (see drawing).

Each of the chains of chemically synthesized oligonucleotides (1-10) is phosphorylated separately. The reaction is carried out in 10 ml of Tris-Hcl pH 9.5 buffer containing 10 mM MgCl ₂ , 50 mM dithiothreitol, 1 mM ATP, 100 pmol oligonucleotide, 1 unit. phage T ₄ polynucleotide kinase for 1 h at 37 ^C. After completion of the reaction the enzyme is inactivated by heating 65 ^{° C} 10 min.

For cloning the somatostatin-14 gene, plasmid vector pBR 325 is used, which contains genes for resistance to ampicillin, tetracycline and chloramphenicol as marker genes. The molecular cloning of the gene encoding somatostatin-14 is carried out as follows: 5 mg of plasmid pBR 325 is incubated with restriction endonucleases EcoRI and BamHI (5 units each) in Tris-HCl buffer, pH 7.5, containing 100 mM NaCl, 7 mM MgCl ₂ , 7 mM 2-mercaptoethanol at ³⁷ ° C for 2 hours. After incubation, samples were heated at 65 ^{° C} for 10 minutes and the fragments were separated by electrophoresis on a 0.8% agarose gel. A large fragment (4042 bp) is extracted from the gel with five volumes of 1.5 M NaCl. The supernatant obtained after centrifugation of the suspension is treated with phenol and chloroform, the DNA is precipitated with ethanol, pre-added t-RNA to a concentration of 10 μg / ml and dissolved in TE buffer (10 mM Tris-Hcl, pH 7.4, 1 mM EDTA).

50 mmol of each phosphorylated oligonucleotide is added to 1 μg of DNA of a large pBR 325 fragment. Ligation of a mixture of oligonucleotides and a DNA fragment is carried out in a medium containing 66 mM Tris-HCl, pH 7.6, 5 mM dithiothreitol, 5 mM MgCl ₂ , 1 mM ATP and 1 unit Phage T ₄ DNA ligases. Incubation is carried out at 12 ^about C for 16 hours

The resulting ligation mixture of DNA fragment and oligonucleotides is introduced by transformation into E. coli HB101 cells. The transformation is carried out using frozen E. coli HB101 cells. 5 fresh colonies of 2 mm are dispersed on a shaker in 1 ml of SOB medium (2% bacto-tryptone, 0.5% yeast extract. 10 mM NaCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ ). 500 .mu.l of the cell suspension is introduced into 10 ml of SOB liquid medium and grown at ³⁷ ° C to a titer of 7x10 ⁷ cells / ml. Cells were cooled on ice and centrifuged at 750 g for 15 min at ⁴ ° C, the supernatant was carefully removed and the pellet resuspended in 3.3 ml buffer with rubidium chloride (100 mM RbCl, 50 mM MgCl ₂ x 4H ₂ O, 30 mM acetate potassium, 10 mM CaCl ₂ x 2H ₂ O, 10 mM glycerol). Cells are kept on ice for 2 hours, centrifuged at 750 g and the supernatant is thoroughly removed, resuspended in 800 μl of buffer containing 10 mM MOPS, 10 mM RbCl, 75 mM CaCl ₂ x 2H ₂ O, 15% glycerol and incubated for 15 min in ice, after whereby aliquots of the final suspension are frozen in liquid nitrogen and used for transformation.

200 l of the prepared cell suspension is mixed with 5-10 l of a solution of the ligated DNA fragment and the mixture of oligonucleotides and incubated on ice, then 30 s at 42 ^{° C} and again in ice for 2 minutes, was added 800 .mu.l of SOC medium (SOB supplemented with 20 mM glucose) and incubated with moderate shaking at 37 ^about C for 60 minutes

 A small portion of the cell suspension is scattered onto agar plates prepared in SOB medium containing 0.8% bacto-agar and 50 μg / ml ampicillin.

 12 clones grown on ampicillin plates were randomly selected, plasmid DNA was isolated by the alkaline method. After treatment of these plasmids with various restriction endonucleases and agarose gel electrophoresis, a plasmid of the desired construction is selected that contains the vector fragment pBR 325 containing the bla gene, which determines resistance to ampicillin, and the somatostatin-14 gene and designate it as pSOM 3-13. The analysis of the nucleotide sequence of the EcoRI - BamHI insert from this plasmid is carried out by the Maxam-Hilbert method and confirm the presence of a sequence encoding somatostatin-14 in it.

 PRI me R 2. Construction of a plasmid for the expression of somatostatin in E. coli cells.

To express somatostatin as part of a chimeric protein in E. coli cells, a recombinant plasmid is constructed in several steps. At the first stage, intermediate plasmids pCMRI ^- and pSOM 3-13-5 are constructed.

A. Construction of the plasmid pCMRI ^- .

 In the pcMRI vector, the genes encoding resistance to ampicillin and chloramphenicol are taken from plasmid pBR 325, and the tandem of phage fd transcription terminators from plasmid pLK 53.

5 μg of plasmid pBR 325 and pBK 53 are treated together with restriction endonucleases PstI and HindIII (10 units each) in 50 μl of buffer containing 10 mM trans HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl ₂ , 1 mM dithiothreitol at 37 ^{° C} for 2 hours, then heated for 5 min at 65 ^C. The fragments are separated on a 0.8% agarose gel electrophoresis. Isolation of a small fragment (1900 bp) of pBR 325 and a large fragment (2240 bp) of pLK 53 is carried out as in example 1.

To combine the fragments obtained in this way, 0.5 μg of DNA are mixed and ligated in a buffer containing 66 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 5 mM dithiothreitol, 1 mM ATP and 1 unit. DNA T4 ligase for 16 h at 12 ^C. The resulting mixture was joined fragments are introduced into cells by transformation E. coli HB 101 calcium method.

50 ul of cell suspension was E. coli HB 101 is introduced into 10 ml of LB liquid medium and the cells grown at ³⁷ ° C to a titer of 7x10 ⁷ cells / ml. Cells were cooled on ice for 10 minutes and centrifuged at 750g for 15 min at 4 ^C. The cell pellet was resuspended in 5 ml cold 0.1 M CaCl ₂ and incubated on ice for 30 min, then the cells were recentrifuged and the pellet resuspended in 0.8 ml of cold 0.1 M CaCl ₂ . Cells are kept in ice for 12 hours, after which they are used for transformation.

To 200 ul of cells were added to 10 .mu.l of the DNA fragments coupled and incubated on ice for 40 minutes, then held at 42 to 90 ^{° C} and rapidly cooled on ice, add 1 ml SOC medium and incubated at ³⁷ ° C for 60 min.

Cells are plated on plates with ampicillin (50 μg / ml) and chloramphenicol (30 μg / ml), and then tested for resistance to tetracycline (20 μg / ml). Plasmid DNA is isolated from clones having the Ap ^r Cm ^r Tc ^s phenotype and analyzed by electrophoretic mobility and restriction analysis. Identify the plasmid with the desired option and designate pCM.

 From the PCM plasmid, the EcoRI restriction enzyme cleavage site located inside the CAT coding sequence is removed to obtain a plasmid convenient for embedding somatostatin-14, flanked by EcoRI - BamHI sites, in the C-terminal region of CAT.

 To remove the EcoRI site, a chemically synthesized linker is used having the sequence: 5'-AATTGGTGG-3 '- one chain (I) and 3'-CCACCTTAA-5' - the second chain (II).

3 ug of plasmid DNA was hydrolyzed PCM EcoRI restriction enzyme in 30 microliters of a solution containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl _2, 1 mM dithiothreitol, 100 mM NaCl at ³⁷ ° C for 2 hours. The reaction mixture was heated 5 min at 65 ^about C, extracted with phenol and DNA precipitated with ethanol. The resulting preparation of a linear form of the vector PCM was dissolved in 30 μl of TE buffer.

50 mmol of phosphorylated cold ATP oligonucleotides (I and II), 1 unit, are added to 3 μl of the pCM vector DNA solution. Phage T ₄ DNA ligases in 30 μl 66 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 5 mM dithiothreitol, 1 mM ATP. The reaction mixture was incubated at 12 ^{° C} for 16 hours.

The resulting ligase mixture was introduced into E. coli HB101 cells by calcium transformation method as in Example 2A. The cell suspension after transformation is plated on agar plates containing ampicillin (50 μg / ml) and chloramphenicol (30 μg / ml). Plasmid DNA is isolated from the clones having the Ap ^r Cm ^r phenotype by the alkaline method, analyzed by electrophoretic mobility and restriction analysis. A plasmid is selected that has lost its cleavage with the restriction enzyme EcoRI and is designated pCMRI ^- .

 B. Construction of the intermediate plasmid pSOM 3-13-5.

 For the construction of plasmids pSOM 3-13-5, plasmids pSOM 3-13 and pRK are used. Plasmid pRK is similar to plasmids of the pUC series, but has the following polylinker nucleotide sequence (9): 5′-AATTCCCGGGAAACTTCGAACGGATCCTGCAGTCGACGGTACCCA-3 ′.

5 μg of plasmid pRK and plasmid pSOM 3-13 are hydrolyzed together with restriction endonucleases ScaI (10 units) and EcoRI (10 units) in 50 μl of Tris-HCl pH 7.5 buffer containing 125 mM NaCl, 7 mM MgCl ₂ , 7 mM 2-mercaptoethanol and 0.01% BSA at ³⁷ ° C for 2 hours. Then, heated at ⁶⁵ ° C for 5 min.

 The large fragment (3529 bp) of the pSOM 3-13 plasmid and the small fragment (957 bp) of the pRK plasmid were isolated from 0.8% agarose gel as in Example 1. DNA was precipitated with ethanol and dissolved in 10 μl of TE buffer.

5 μl of solutions of the obtained fragments are mixed and ligation is carried out in a buffer containing 66 mM Tris-HCl, pH 7.6, 5 mM dithiothreitol, 5 mM MgCl ₂ , 1 mM ATP and 1 unit. Phage T ₄ DNA ligases. Incubation was carried out at 12 ^{° C} for 16 hours. The ligated mixture of DNA fragments is introduced into cells of E. coli HB101 transformation method described in Example 2A.

 The selection of recombinant clones is carried out after plating a suspension of cells on plates with ampicillin (50 μg / ml). Plasmids are isolated from clones, and the products of their hydrolysis obtained by the combined action of EcoRI and ScaI restriction enzymes are analyzed. A plasmid having the desired construct is selected and designated as pSOM 3-13-5.

 B. Construction of the target plasmid.

From the plasmid pCMRI ^- (example 2A) and plasmid pSOM 3-13-5 (example 2B), the target plasmid is constructed.

From the plasmid pCMRI ^- , which has two sites of action of the ScaI restrictase, a small fragment (1389 bp) containing the CAT gene shortened from the 3'-end is cleaved. Plasmid pSOM 3-13-5 is digested with restriction enzymes ScaI and SmaI and a large fragment (3531 bp) carrying the somatostatin-14 gene is isolated. The isolated fragments are mixed and ligated, repeating all the conditions for plasmid DNA fragment isolation and ligation as in Example 1. The ligation mixture of DNA fragments is introduced by transformation into E. coli HB101 cells using the transformation method as in Example 1. Cells are seeded on plates with ampicillin (50 μg / ml). From clones grown on ampicillin plates, a plasmid with the desired construction is isolated containing the CAT-somatostatin-14 hybrid gene and is designated pCCS.

 The nucleotide sequence is analyzed in the region of recognition by the EcoRI restriction enzyme according to the Maxam-Gilbert method, which shows that the linked cleavage sites of the ScaI restriction enzyme located at the 3'-end of the CAT gene with the Smal site from the polylinker of the pRK plasmid allow one to obtain a single translation phase upon expression of the hybrid gene CAT-somatostatin-14.

 PRI me R 3. The use of recombinant plasmids pCCS to create a strain of E. coli - producer of somatostatin-14.

 Plasmid pCCS was introduced by transformation into E. coli MKD 3207 strain according to the method described in example 1, and a sommatostatin-14 producing strain was obtained.

 Strain MKD 3207 is characterized by the following features. Cultural and morphological.

Strain MKD 3207 (Escherichia coli K 12) - gram-negative, sedentary sticks, under adverse conditions forming filaments. The strain grows well in a temperature range of 30-42 ^{° C} in rich media type Hottinger agar and broth, as well the BCH and IPA, and synthetic media supplemented with compensating auxotrophic mutation.

On rich media, colonies are formed that are smooth, with smooth edges, which mucilize over time, due to the lon mutation. At a temperature of 40-42 ^{° C} incubation, colonies mucilaginized occurs. When growing on a synthetic medium with additives, the colonies are always mucous.

 Genetic and physiological-biochemical.

Strain MKD 3207 has the following genetic markers: F ^- lac Y, sup E, gal 6, xyl 4, mal A1, arg H, his', lon ^- , apr 24, rpl and is of the female type. Resistant to streptomycin, does not ferment lactose, galactose, xylose and maltose.

 The strain grows on a synthetic medium supplemented with glucose, arginine and histidine.

 Strain MKD 3207, containing the plasmid pCCS, becomes resistant to ampicillin.

The expression of the gene encoding somatostatin in the fusion protein in E. coli MKD 3207 cells is analyzed. The fusion gene in the pCCS expression vector in E. coli MKD 3207 cells directs the constitutive synthesis of the fusion protein under the control of its own CAT-P _cat promoter.

Cells E. coli MKD 3207 transformed with plasmid pCCS, grown in medium LB, containing ampicillin (50 ug / ml) to OD ₆₆₀ 1.8-2.0 density at ³⁷ ° C for 18 hours. As a control vector plasmid pBR 325 and plasmid pCMRI ^- encoding the native CAT protein under the control of the P _cat own promoter, as in the fusion protein. From 1.5 ml of cell culture, a cell pellet is obtained by centrifugation. The precipitate was suspended in 20 μl of a buffer solution containing 50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 2% 2 mercaptoethanol. The suspension is boiled for 5 minutes and analyzed by electrophoresis in 15% SDS PAGE. The results show the presence of a dominant band in the molecular weight region of 24 kD. The expression level of the hybrid protein is 5% of the total proteins of the bacterial cell.

 PRI me R 4. Identification of somatostatin in E. coli cells. A. Isolation of the fusion protein.

Cells E. coli MKD 3207 transformed with plasmid pCCS, cultured in LB medium containing ampicillin (50 ug / ml) at 37 ^{° C} for 6-7 h to 10 l fermenter density OD ₅₅₀ of 2.0-2.5. Cells are pelleted by centrifugation at 5000 g for 10 minutes.

The cell pellet was suspended in Tris-HCl buffer pH 8.0, containing 100 mM NaCl and 1 mM EDTA at the rate of 38 ml of buffer per 1 L of cell culture. Lysozyme is added to a final concentration of 0.2 mg / ml and the suspension is incubated in ice. The extract was frozen and thawed at ³⁷ ° C in a water bath. Cells are destroyed by ultrasound. The precipitate, comprising insoluble fusion protein is obtained by centrifugation 1200g for 5 minutes at ⁴ ° C, washed in buffer with 0.5% Triton X-100 (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 mM NaCl, 0,5 % Triton X-100), centrifuged again and the pellet resuspended in the original buffer. Aliquots of 10-15 μl were selected and analyzed by 15% SDS-PAGE by electrophoresis. Discover a dominant band in the area of the pier. m. 24 kD corresponding to the hybrid protein and constituting 70-80% of the total protein of the sediment.

 B. Obtaining somatostatin.

The fusion protein purified according to Example 4A was dissolved in 0.2 M Tris-HCl pH 8.0 buffer containing 6 M guanidine chloride and 2 mM EDTA. A 50-fold molar excess of dithiothreitol per SS group of the fusion protein was added and the solution was dialyzed overnight against 0.01 M NH ₄ HCO ₃ . The resulting precipitate was separated by centrifugation fusion protein 1200g for 15 min at 4 ^{° C} and used for cleavage with cyanogen bromide.

 The hybrid protein precipitate was dissolved in 70% formic acid and crystalline cyanogen bromide (2 mg / mg protein) was added. Incubation is carried out at room temperature for 20 hours. The completeness of hydrolysis is monitored using 9-26% SDS-PAGE electrophoresis.

 The mixture of peptides formed after cleavage with cyanogen bromide is fractionated on Sephadex G-50. The identification of somatostatin in the fractions combined according to the chromatographic profile after their preliminary desalination is carried out using radioimmunological analysis using Incstar kits (USA). Fractions containing somatostatin are purified by reverse phase chromatography using a Diasorb C 16 column (4x50 mm, grain size 8 μm).

 Peptides having somatostatin immunoreactivity are collected, frozen, freeze-dried and used to determine the N-terminal amino acid using dansyl chloride and amino acid analysis. The results of amino acid analysis are presented in the table.

 The output of somatostatin is 1 mg per 1 liter of bacterial culture. The immunochemical purity of the drug according to radioimmunological analysis exceeds 85% and corresponds to the commercial drug company "Sigma" (USA).

(56) 1. ЕР N 0001929, кл. C 12 N 15/00, 1979.

(56) 1. EP N 0001929, cl. C 12 N 15/00, 1979.

 2. EP N 0160190, class C 12 N 15/00, 1986.

 3. EP N 0108045, class C 12 N 15/00, 1983.

 4. EP N 0036776, cl. C 12 N 15/00, 1981.

 5. EP N 0197558, cl. C 12 N 15/00, 1986.

 6. Patent US N 4426437, cl. 435/317, 1984.

 7. CH 662128, cl. C 12 N 15/00, 1987.

Claims (2)

1. Recombinant plasmid DNA pccS encoding somatostatin-14, size 4920 p. N. containing:
- a fragment of plasmid vector pBR 325 with a size of 4860 bp comprising a part of the tetracycline resistance gene with the Bam HI restriction site at the 5'-end, an ampicillin resistance gene, a part of the chloramphenicolacetyltransferase gene with a restriction half site
ScaI at the 3'-end and the eliminated restriction site Eco RI;
- a synthetic somatostatin gene with a stop codon flanked by Eco RI - Bam HI restriction sites, 58 bp in size. ;
- a linker fragment containing the Eco RI restriction site and flanked from the 5'-end of the GG nucleotide sequence for fusion of the somatostatin gene with
3'-end of the chloramphenicol acetyltransferase gene;
- genetic markers:
- Ap ^r gene - ampicillin resistance gene;
- unique restriction sites located clockwise from the Bam HI site: Bam HI, Sal I, Sph I, Nru I, Ava I, Nde I, Ssp I, Pst I, Pm I, Sca I, Eco 0109, Nco I, Eco RI.  2. The bacterial strain Escherichia coli VKPM B 5557 - producer of somatostatin-14.

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