PL229055B1 - DNA sequence, oligonucleotide primers, DNA sequence with the recognition sites for restriction enzymes NdeI and XhoI, expression vector and method for the preparation thereof, recombinant strain of Escherichia coli Rosetta (DE3) pLysS and method for the preparation thereof and method for obtaining recombinant protein with oligo-histidine tag of Deinococcus radiodurans DSMZ 20 539 trehalose synthase (TS) - Google Patents

Description

The present invention relates to a method for the preparation of a recombinant Escherichia coli Rosetta (DE3) pLysS strain containing cloned DNA with a fragment encoding the Deinococcus radiodurans trehalose synthase protein DSMZ 20539 and a method for the production of Deinococcus radiodurans trehalose synthase protein DSMZ 20539.

Trehalose (α-D-glucopyranosyl-1,1a-D-glucopyranoside) is a non-reducing sugar composed of two glucose molecules linked by an α-1,1-glycosidic bond. Trehalose is easily hydrolyzed to glucose and can fill the cell's reserve of this sugar. The occurrence of trehalose was found in the cells of fungi and yeasts, bacteria, nematodes, insects, eggs, pupae and some plants. The properties of trehalose make it a potential source of many applications in human, veterinary, pharmaceutical and food industries.

Trehalose biosynthesis in living cells is carried out by various known metabolic pathways, including the use of trehalose synthase.

Bacteria of the genus Deinococcus belong to the microorganisms that produce trehalose synthase.

The genes encoding the trehalose synthase proteins of microorganisms of the genus Deinococcus were cloned in E. coli cells. The biosynthesis of thermostable enzymes in mesophilic host cells was also carried out in the Tabor-Studier system. Unfortunately, the E. coli expression systems used and with the use of the expensive inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) are not optimal and the most efficient. Moreover, the obtained proteins did not have the oligohistidine tag which would significantly facilitate the process of obtaining the oligohistidine tag, and the preliminary thermal denaturation of the host proteins was not used during the purification procedure itself.

In the course of the work, the isolation and cloning of the gene encoding the Deinococcus radiodurans trehalose synthase into expression vectors in the Tabora-Studier and arabinose systems were performed, and its expression in various hosts was checked. The optimal purification process was also established and the properties of DraST wild-type recombinant proteins in the arabinose system and with the DraSTHis oligohistidine domain were determined.

Protein purification is based on the innovative application of pre-denaturation of host proteins and metal affinity chromatography.

A high purity preparation was obtained for the DraSTHis protein. The optimal operating conditions for both DraST and DraSTHis proteins have been established. The temperature range from 5 to 20 ° C and pH 6.5 were determined as the optimal conditions for the enzyme operation.

Molecular filtration allowed the determination of the molecular weight of the DraSTHis protein, which is 126.9 kDa, which corresponds to the mass of the homodimer.

The method of obtaining the recombinant Escherichia coli Rosetta (DE3) pLysS strain, symbol Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH, is characterized according to the invention in that a DNA fragment encoding the Deinococcus radiodurans trehalose synthase protein Deinococcus radiodurans DSMZ 20539 of the formula 3 is obtained. using oligonucleotide primers of formula 2 and symbols DraSTNdeF and DraSTXhoR. The obtained DNA fragment is cloned into the pET30Ek / LIC plasmid vector, as a result of which the plasmid vector of formula 4 and the symbol pET30Ek // LICDraSTH is obtained, containing the cloned DNA with the fragment encoding the Deinococcus radiodurans trehalose synthase protein DSMZ 20539, with fragments recognized by restriction enzymes and XhoI, with the rbs ribosome binding site, with the kanamycin resistance gene, with the origin of the pBR322 plasmid, with the origin f1, the polyhistidine region, the T7 promoter, the S-Tag coding sequence, with the T7 transcription termination site, with the lacI operator site. Then Escherichia coli Rosetta (DE3) pLysS cells were transformed with the obtained vector.

The method for producing the Deinococcus radiodurans DSMZ 20539 trehalose synthase protein consisting in the production of the protein by cells of a recombinant bacterial strain in liquid media, is characterized according to the invention in that cells of the Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH bacterial strain are cultivated at a temperature of starting from 35 ° C to 38 ° C. Then, after obtaining a density of the cell suspension from 0.3 to 0.9, overexpression of the gene encoding the protein is carried out for 2 to 24 hours by adding an inducer, which is IPTG, with a concentration of 0.05 mM to 10 mM to the medium, and after separating the suspension from the culture fluid, cell disintegration and initial thermal denaturation, the soluble proteins are isolated by centrifugation. They are then purified using one purification step on IDA-Co coordinated reversible metal ion-binding beds, then concentrated and suspended in storage buffer.

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The storage buffer preferably has the following composition: 0.85% NaCl, 20% glycerol (v / v), 5mM EDTA, 1mM DTT, 1mM PMSF, 0.05% IGEPAL.

The recombinant Deinococcus radiodurans DSMZ 20539 trehalose synthase protein obtained according to the invention is characterized by high activity at room and sub-room temperatures, which allows it to be used in the continuous conversion of maltose into trehalose without the need to incur expenditure on heating the column bioreactor.

The method according to the invention makes it possible to obtain the recombinant Deinococcus radiodurans DSMZ 20539 trehalose synthase protein by a fast method characterized by a limited number of steps needed to obtain a protein preparation.

The invention is explained in more detail in the working examples and in the drawings, in which Fig. 1 shows the DNA fragment of the formula 3 with the sequence of the Deinococcus radiodurans DSMZ 20539 trehalose synthase gene and DNA fragments recognized by the restriction enzymes NdeI and XhoI;

Figure 2 shows the nucleotide sequence of the pET30Ek / LICDraSTH plasmid vector in the cloning region of the DNA fragment of formula 3 and the amino acid sequence of the protein produced in Escherichia coli Rosetta (DE3) pLysS cells transformed with pET30Ek / LICDraSTH expression vector DNA;

Figure 3 shows the result of the electrophoretic separation on a 12% polyacrylamide gel with a 6% thickening layer of fractions from the subsequent isolation and purification steps of the Deinococcus radiodurans trehalose synthase protein DSMZ 20539 produced by the recombinant Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH strain;

Figure 4 shows a scheme for obtaining a DNA fragment of formula 3 with the sequence of the Deinococcus radiodurans DSMZ 20539 trehalose synthase gene and DNA fragments recognized by the restriction enzymes NdeI and XhoI and the expression vector of formula 4 and the symbol pET30Ek / LICDraSTH.

P r z k ł a d 1

Preparation of DNA sequence encoding Deinococcus radiodurans trehalose synthase protein DSMZ 20539.

For the amplification of DNA encoding the Deinococcus radiodurans DSMZ 20539 trehalose synthase protein, oligonucleotide primers of the formula 2 and the symbols DraSTNdeF and DraSTXhoR are synthesized in a known manner. The amplification reaction of the DNA encoding the Deinococcus radiodurans DSMZ 20539 trehalose synthase protein is performed using the primers DraSTNdeF (0.2 μM) and DraSTXhoR (0.2 μM) on the template of the isolated genomic DNA in a buffer containing: 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH4) 2SO4, 2 mM MgSO4, 0.1% Triton X-100, 200 μM of each deoxyribonucleoside triphosphate (dNTP) and 0.2 U of Pwo DNA polymerase produced by DNA-Gdańsk II sc, Gdańsk , Poland. The reaction is carried out in a thermal cycler under the following temperature-time conditions: 95 ° C - 1 minute, 63.5 ° C - 1.5 minutes, 72 ° C - 2 minutes; 35 cycles. The amplification reaction yields a DNA fragment of formula 3. A DNA fragment of formula 3 with the exact sequence of the gene encoding the Deinococcus radiodurans DSMZ 20539 treahlose synthase protein and DNA fragments recognized by the restriction enzymes NdeI and XhoI are shown in Fig. 1.

P r z k ł a d 2

Preparation of the expression vector of formula 4 and symbol pET30Ek / LICDraSTH and recombinant Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH strain.

The scheme of obtaining the expression vector of formula 4 and the symbol pET30Ek / LICDraSTH is shown in Fig. 4. In order to obtain the plasmid vector pET30Ek / LICDraSTH, the DNA fragment of formula III obtained according to example 1 is cloned into the pET30Ek / LIC expression vector from Merck. For this purpose, a DNA fragment of 5,234 bp resulting from digestion of the Merck plasmid pET30Ek / LIC with restriction enzymes NdeI and XhoI (vector) is carried out with a DNA fragment of 1658 bp resulting from digestion of the PCR product of formula 3 with restriction enzymes NdeI and XhoI (insert) at 17 ° C for 3 hours with 2 U of T4 DNA ligase using reaction buffer: 33 mM Tris-CH3COOH pH = 7.8; 10mM (CH3COO) 2Mg; 66 mM CH3COOK; 0.5 mM DTT; 1 mM ATP.

Then the reaction mixture is transformed into Escherichia coli Rosetta (DE3) pLysS competent cells and, after shaking for 1 h at 37 ° C, about 230 rpm, in 250 μl of LB medium, it is plated on LA medium (LB medium supplemented with 1.5 % agar according to Sambrook et al., 1989), containing tetracycline and kanamycin.

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The resulting Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH bacterial colonies containing plasmid expression vectors are screened on LB medium supplemented with tetracycline and kanamycin and grown to isolate plasmid DNA.

As a result of the isolation, DNA of the plasmid vector of formula 4 and symbol pET30Ek / LICDraSTH was obtained, which contains the cloned gene encoding the Deinococcus radiodurans trehalose synthase protein DSMZ 20539 under the control of the strong T7 phage promoter (Fig. 2), which was confirmed by restriction analysis and DNA sequencing , e.g. by the Sanger method.

The exact nucleotide sequence of the plasmid vector of formula 4 and the symbol pET30Ek / LICDraSTH in the cloning region of the DNA fragment of formula 3 is illustrated in Figure 2.

P r z k ł a d 3

Preparation of Deinococcus radiodurans trehalose synthase protein DSMZ 20539.

The DNA sequence encoding the Deinococcus radiodurans trehalose synthase protein DSMZ 20539 is obtained along with the recognition sites for the restriction enzymes NdeI and XhoI of Formula 3 according to Example 1.

The expression vector plasmid of formula 4 and the symbol pET30Ek // LICDraSTH is then obtained according to example 2.

In the next step, the recombinant Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH strain is obtained by transforming Escherichia coli Rosetta (DE3) pLysS competent cells and selective pressure on chloramphenicol and kanamycin media.

The strain is cultivated in LB medium supplemented with ampicillin for 18 hours at 37 ° C. An overnight culture of the recombinant E. coli strain is inoculated with 500 ml of LB medium with antibiotic, and after the culture has reached a cell suspension density of OD 600nm> 0.5, expression of IPTG trehalose synthase protein is induced. Cultures 3.5 hours after induction performed at room temperature (500 ml medium volume) are centrifuged for 10 minutes at 5000 rpm. The bacterial pellet is suspended in 30 ml of lysis buffer, homogenized and sonicated for 10 s - at amplitude A = 30%, repeating the procedure three times at short intervals. The resulting total lysate is centrifuged for 40 minutes at 9,000 rpm. Deinococcus radiodurans DSMZ 20539 trehalose synthase protein is purified by thermal precipitation of other proteins present in the preparation (mesophilic E. coli protein) at 56 ° C for 10 minutes. The lysate is centrifuged for 30 minutes at 12,000 rpm. The supernatant containing undenatured soluble proteins is further purified using metal affinity chromatography. In another variant of the purification, the proteins obtained can be subjected to precipitation with ammonium sulphate or precipitation with ethanol, acetone, isopropanol or other low-alcohol alcohol.

The IDA-Co matrix column is calibrated with 5 bed volumes of buffer A: 5 mM imidazole, 50 mM phosphate buffer, 50 mM NaCl, pH 7.6. Then, an amount of total cell-free extract equal to the volume of the bed is applied to the bed. The column is washed with low salt A buffer: 5 mM, 50 mM phosphate buffer, 50 mM NaCl and A 'at 25 mM imidazole, 50 mM phosphate buffer, 50 mM NaCl at pH = 7.6 to remove unbound proteins. DraST is eluted with two 10 ml aliquots of high-salt phosphate buffer with pH = 7.6 and an imidazole concentration of 500 mM, 50 mM phosphate buffer, 50 mM NaCl.

The preparation after purification on the IDA-Co column should be centrifuged three times on centrifuge filters with a cut-off of 30 kDa (Amicon, Millipore), at 5000 rpm, for 30 minutes, successively adding the elution fractions obtained above to concentrate the protein. During concentration, the high-salt buffer should be replaced with a buffer for storing the protein preparation (e.g. containing protease inhibitors or glycerol and / or saline. Preferably, the composition consists of: 0.85% NaCl, 20% glycerol (v / v), 5 mM EDTA , 1 mM DTT, 1 mM PMSF, 0.05% IGEPAL. In another variant, the obtained protein preparation can be dialyzed into the desired buffer. It is also possible to use the total cell-free extract for the conversion of maltose to trehalose after the lysis step or only after the initial thermal denaturation of the proteins the host.

The results of the biosynthesis and purification of Deinococcus radiodurans DSMZ 20339 trehalose synthase protein are analyzed by electrophoresis on a 12% polyacrylamide sol under denaturing conditions in the presence of an appropriate size marker protein as shown in Figures 3 and 3a.

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Formula 1. Sequences of the gene encoding the Deinococcus radiodurans trehalose synthase protein DSMZ 20539

ATGACCCAGGCACACCCGGAGTGGTACAAGAGCGCTGTCTTCTACGAACTGTCCGTGC

GGACCTTTCAGGACGGCAACGGCGACGGCAAGGGCGATTTTCCCGGCCTGACCTCGC

GGCTGGACTACCTGAAAAACCTCGGCGTGGACTGCCTGTGGTTGCTGCCCTGGTTTCC

CAGCCCACTGCGCGACGACGGGTACGACGTGGCCGACTACCGGGGGATTCACCCTGA

CCTCGGCACGCTCGACGACTTCAAGGTCTTTCTGCGCGAAGCCCACGCCCGGGGCCTG

CGGGTCATCGGCGACCTGGTGACCAACCACACGTCCAGCGACCACCCCTGGTTTCAGG

CGGCGCGGCGCGGCCCGACCCTGCCCGACGGCTCGCCCAACGAGTACCACGACTACT

ACGTCTGGAGCGACGAGGGCAAGGAATACGCCGACACCCGCATCATCTTCACCGACA

CCGAGGTCAGCAACTGGACGCTCGACGAGCAGGCAGGCAAGTATTACTGGCACCGCT

TTTTCGCCAGCCAGCCGGACCTGAACTACGACAACCCGAAAGTGGTGGAAGAACTTC

ACGGCGCCGCCCGCTTCTGGCTCGACCTCGGCCTCGACGGGTTCCGGGTGGACGCCGT

GCCCTACCTCATCGAGCGCGAGGGCACGAGCTGCGAGAATCTGCCCGAAACACACGA

GATTCTCAAGGGCTTCCGGGCGATGGTGGACCGCGAGTATCCGGGGCGGCTGCTGCTT

GCCGAGGCGAACCAGTGGCCCGAGGAAGTCGTCGAGTACTTCGGCACCGAAGCCGAG

CCCGAGTTCCACATGTGCTTCAACTTCCCGGTGATGCCCCGGCTGTACATGAGCCTGA

AAAGGGAGGACACCTCCAGCATCCGCGAGATCATGGGCCGCCTGCCGAAAATCCCGA

GTTTCGGCCAGTGGTGCACCTTCCTGCGCAACCACGACGAACTGACGCTGGAAATGGT

CACCGACGACGAGCGCGCCTTCATGTATGCCGCCTACGCGCCCGACGCCCGCATGAA

AATCAACGTGGGCATCCGCCGTCGCCTCGCGCCGCTGCTCGACAACGACCGGCGCCG

CATCGAGCTGCTGAACACTGTGCTCCTCGCCCTGCCCGGCAGCCCCATCCTGTACTAC

GGCGACGAAATCGGCATGGGCGACGACCTCGGCCTGCCCGACCGCAACGGCGTGCGC

ACCCCGATGCAGTGGAACGCGGGCACCAGCGGCGGTTTTTCCACTGCGCAGCCCTCCG

ACTGCTTTTTCCCGCCGATTCAGGACCCGGTGTACGGCTTCGGGCGCGTCAACGTGCA

GAGCCAGCTCCAAGACCCCAGCAGCCTGCTGAAGTGGACCGCCCGGCAACTCGAACT

GCGCCGCGCCCACCCCGCCTTTGCGCACGGCGACCTCACCTTCATCGAAACCGGCAAC

CCCGCCATCCTCGCCTTTACCCGCCAGTACGACGGCGAAACGCTGCTCATCGTCAGCA

ACTTCGCGGGCAACGCCCAGGCCGGGCTGCTCGATCTCGCGCCCTTCGTGGGCCGCGC

CCCGGTCACACTTTCCGGGGCCAGCCCGCTGCCGGTGGTCACTGGAAACGGCCAGTAC

CCGGTGGTGATGGGCAAGTACGACTATTACTGGCTGCGGTTGAATTGA

Sequence information:

LENGTH: 1659 nucleotides

TYPE: nucleic acid

TOPOLOGY: linear

THREAD COUNT: one

TYPE OF PARTICLE: DNA

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Formula 2. Primer sequences

DraSTNdeF

5 'aa cat ATG ACC CAG GCA CAC CCG GA 3'

Sequence information:

LENGTH: 25 nucleotides

TYPE: nucleic acid

TOPOLOGY: linear

TYPE OF PARTICLE: DNA

DraSTXhoR

5 'aa ctc gag ATT CAA CCG CAG CC A GTA AT A GTC 3'

Sequence information:

LENGTH: 32 nucleotides

TYPE: nucleic acid

TOPOLOGY: linear

TYPE OF PARTICLE: DNA

Formula 3. Sequence of the amplified gene encoding the protein Deinococcus radiodurans DSMZ 20539 along with additional recognition sites for the restriction enzymes NdeI and XhoI.

AACATATGACCCAGGCACACCCGGAGTGGTACAAGAGCGCTGTCTTCTACGAACTGT

CCGTGCGGACCTTTCAGGACGGCAACGGCGACGGCAAGGGCGATTTTCCCGGCCTGA

CCTCGCGGCTGGACTACCTGAAAAACCTCGGCGTGGACTGCCTGTGGTTGCTGCCCTG

GTTTCCCAGCCCACTGCGCGACGACGGGTACGACGTGGCCGACTACCGGGGGATTCA

CCCTGACCTCGGCACGCTCGACGACTTCAAGGTCTTTCTGCGCGAAGCCCACGCCCGG

GGCCTGCGGGTCATCGGCGACCTGGTGACCAACCACACGTCCAGCGACCACCCCTGG

TTTCAGGCGGCGCGGCGCGGCCCGACCCTGCCCGACGGCTCGCCCAACGAGTACCAC

GACTACTACGTCTGGAGCGACGAGGGCAAGGAATACGCCGACACCCGCATCATCTTC

ACCGACACCGAGGTCAGCAACTGGACGCTCGACGAGCAGGCAGGCAAGTATTACTGG

CACCGCTTTTTCGCCAGCCAGCCGGACCTGAACTACGACAACCCGAAAGTGGTGGAA

G AACTTC A CGGCGCCG C CCGCTTCTGGCTCGA CCTCGG CCTCGACGGGTTCCGGGTGG

ACGCCGTGCCCTACCTCATCGAGCGCGAGGGCACGAGCTGCGAGAATCTGCCCGAAA

CACACGAGATTCTCAAGGGCTTCCGGGCGATGGTGGACCGCGAGTATCCGGGGCGGC

TGCTGCTTGCCGAGGCGAACCAGTGGCCCGAGGAAGTCGTCGAGTACTTCGGCACCG

AAGCCGAGCCCGAGTTCCACATGTGCTTCAACTTCCCGGTGATGCCCCGGCTGTACAT

GAGCCTGAAAAGGGAGGACACCTCCAGCATCCGCGAGATCATGGGCCGCCTGCCGAA

AATCCCGAGTTTCGGCCAGTGGTGCACCTTCCTGCGCAACCACGACGAACTGACGCTG

GAAATGGTCACCGACGACGAGCGCGCCTTCATGTATGCCGCCTACGCGCCCGACGCC

CGCATGAAAATCAACGTGGGCATCCGCCGTCGCCTCGCGCCGCTGCTCGACAACGAC

CGGCGCCGCATCGAGCTGCTGAACACTGTGCTCCTCGCCCTGCCCGGCAGCCCCATCC

TGTACTACGGCGACGAAATCGGCATGGGCGACGACCTCGGCCTGCCCGACCGCAACG

GCGTGCGCACCCCGATGCAGTGGAACGCGGGCACCAGCGGCGGTTTTTCCACTGCGC

AGCCCTCCG A CTGCTTTTTCCCGCCG ATTC AGG ACCCGG TG TACGGCTTCGGGCGCGT

CAACGTGCAGAGCCAGCTCCAAGACCCCAGCAGCCTGCTGAAGTGGACCGCCCGGCA

ACTCGAACTGCGCCGCGCCCACCCCGCCTTTGCGCACGGCGACCTCACCTTCATCGAA

ACCGGCAACCCCGCCATCCTCGCCTTTACCCGCCAGTACGACGGCGAAACGCTGCTCA

TCGTCAGCAACTTCGCGGGCAACGCCCAGGCCGGGCTGCTCGATCTCGCGCCCTTCGT

GGGCCGCGCCCCGGTCACACTTTCCGGGGCCAGCCCGCTGCCGGTGGTCACTGGAAA

CGGCCAGTACCCGGTGGTGATGGGCAAGTACGACTATTACTGGCTGCGGTTGAATCTC

GAGTT

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Sequence information:

LENGTH: 1669 nucleotides

TYPE: nucleic acid

TOPOLOGY: linear

THREAD COUNT: one

TYPE OF PARTICLE: DNA

Formula 4. Sequence of the recombinant pETSOEk / LICDraSTH plasmid.

ATCCGGATAT AGTTCCTCCT TTCAGCAAAA AACCCCTCAA GACCCGTTTA

GAGGCCCCAA

GGGGTTATGC TAGTTATTGC TCAGCGGTGG CAGCAGCCAA CTCAGCTTCC

TTTCGGGCTT 121 TGTTAGCAGC CGGATCTCAG TGGTGGTGGT GGTGGTGCTC GAGATTCAAC CGCAGCCAGT 181 AATAGTCGTA CTTGCCCATC ACCACCGGGT ACTGGCCGTT TCCAGTGACC ACCGGCAGCG 241 GGCTGGCCCC GGAAAGTGTG ACCGGGGCGC GGCCCACGAA GGGCGCGAGA TCGAGCAGCC 301 CGGCCTGGGC GTTGCCCGCG AAGTTGCTGA CGATGAGCAG CGTTTCGCCG TCGTACTGGC 361 GGGTAAAGGC GAGGATGGCG GGGTTGCCGG TTTCGATGAA GGTGAGGTCG CCGTGCGCAA 421 AGGCGGGGTG GGCGCGGCGC AGTTCGAGTT GCCGGGCGGT CCACTTCAGC AGGCTGCTGG 481 GGTCTTGGAG CTGGCTCTGC ACGTTGACGC GCCCGAAGCC GTACACCGGG TCCTGAATCG 541 GCGGGAAAAA GCAGTCGGAG GGCTGCGCAG TGGAAAAACC GCCGCTGGTG CCCGCGTTCC 601 ACTGCATCGG GGTGCGCACG CCGTTGCGGT CGGGCAGGCC GAGGTCGTCG CCCATGCCGA 661 TTTCGTCGCC GTAGTACAGG ATGGGGCTGC CGGGCAGGGC GAGGAGCACA GTGTTCAGCA 721 GCTCGATGCG GCGCCGGTCG TTGTCGAGCA GCGGCGCGAG GCGACGGCGG ATGCCCACGT 781 TGATTTTCAT GCGGGCGTCG GGCGCGTAGG CGGCATACAT GAAGGCGCGC TCGTCGTCGG 841 TGACCATTTC CAGCGTCAGT TCGTCGTGGT TGCGCAGGAA GGTGCACCAC TGGCCGAAAC 901 TCGGGATTTT CGGCAGGCGG CCCATGATCT CGCGGATGCT GGAGGTGTCC TCCCTTTTCA 961 GGCTCATGTA CAGCCGGGGC ATCACCGGGA AGTTGAAGCA CATGTGGAAC

TGGGGCTCGG

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1021 CTTCGGTGCC GAAGTACTCG ACGACTTCCT CGGGCCACTG GTTCGCCTCG GCAAGCAGCA 1081 GCCGCCCCGG ATACTCGCGG TCCACCATCG CCCGGAAGCC CTTGAGAATC TCGTGTGTTT 1141 CGGGCAGATT CTCGCAGCTC GTGCCCTCGC GCTCGATGAG GTAGGGCACG GCGTCCACCC 1201 GGAACCCGTC GAGGCCGAGG TCGAGCCAGA AGCGGGCGGC GCCGTGAAGT TCTTCCACCA 1261 CTTTCGGGTT GTCGTAGTTC AGGTCCGGCT GGCTGGCGAA AAAGCGGTGC CAGTAATACT 1321 TGCCTGCCTG CTCGTCGAGC GTCCAGTTGC TGACCTCGGT GTCGGTGAAG ATGATGCGGG 1381 TGTCGGCGTA TTCCTTGCCC TCGTCGCTCC AGACGTAGTA GTCGTGGTAC ICGTTGGGCG 1441 AGCCGTCGGG CAGGGTCGGG CCGCGCCGCG CCGCCTGAAA CCAGGGGTGG TCGCTGGACG 1501 TGTGGTTGGT CACCAGGTCG CCGATGACCC GCAGGCCCCG GGCGTGGGCT TCGCGCAGAA 1561 AGACCTTGAA GTCGTCGAGC GTGCCGAGGT CAGGGTGAAT CCCCCGGTAG TCGGCCACGT 1621 CGTACCCGTC GTCGCGCAGT GGGCTGGGAA ACCAGGGCAG CAACCACAGG CAGTCCACGC 1681 CGAGGTTTTT CAGGTAGTCC AGCCGCGAGG TCAGGCCGGG AAAATCGCCC TTGCCGTCGC 1741 CGTTGCCGTC CTGAAAGGTC CGCACGGACA GTTCGTAGAA GACAGCGCTC TTGTACCACT 1801 CCGGGTGTGC CTGGGTCATA TGTATATCTC CTTCTTAAAG TTAAACAAAA TTATTTCTAG 1861 AGGGGAATTG TTATCCGCTC ACAATTCCCC TATAGTGAGT CGTATTAATT TCGCGGGATC 1921 GAGATCGATC TCGATCCTCT ACGCCGGACG CATCGTGGCC GGCATCACCG GCGCCACAGG 1981 TGCGGTTGCT GGCGCCTATA TCGCCGACAT CACCGATGGG GAAGATCGGG CTCGCCACTT 2041 CGGGCTCATG AGCGCTTGTT TCGGCGTGGG TATGGTGGCA GGCCCCGTGG

CCGGGGGACT

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2101 GTTGGGCGCC ATCTCCTTGC ATGCACCATT CCTTGCGGCG GCGGTGCTCA ACGGCCTCAA 2161 CCTACTACTG GGCTGCTTCC TAATGCAGGA GTCGCATAAG GGAGAGCGTC GAGATCCCGG 2221 ACACCATCGA ATGGCGCAAA ACCTTTCGCG GTATGGCATG ATAGCGCCCG GAAGAGAGTC 2281 AATTCAGGGT GGTGAATGTG AAACCAGTAA CGTTATACGA TGTCGCAGAG TATGCCGGTG 2341 TCTCTTATCA GACCGTTTCC CGCGTGGTGA ACCAGGCCAG CCACGTTTCT GCGAAAACGC 2401 GGGAAAAAGT GGAAGCGGCG ATGGCGGAGC TGAATTACAT TCCCAACCGC GTGGCACAAC 2461 AACTGGCGGG CAAACAGTCG TTGCTGATTG GCGTTGCCAC CTCCAGTCTG GCCCTGCACG 2521 CGCCGTCGCA AATTGTCGCG GCGATTAAAT CTCGCGCCGA TCAACTGGGT GCCAGCGTGG 2581 TGGTGTCGAT GGTAGAACGA AGCGGCGTCG AAGCCTGTAA AGCGGCGGTG CACAATCTTC 2641 TCGCGCAACG CGTCAGTGGG CTGATCATTA ACTATCCGCT GGATGACCAG GATGCCATTG 2701 CTGTGGAAGC TGCCTGCACT AATGTTCCGG CGTTATTTCT TGATGTCTCT GACCAGACAC 2761 CCATCAACAG TATTATTTTC TCCCATGAAG ACGGTAGGCG ACTGGGCGTG GAGCATGTGG 2821 TCGCATTGGG TCACCAGCAA ATCGCGCTGT TAGCGGGCCC ATTAAGTTCT GTCTCGGCGC 2881 GTCTGCGTCT GGCTGGCTGG CATAAATATC TCACTCGCAA TCAAATTCAG CCGATAGCGG 2941 AACGGGAAGG CGACTGGAGT GCCATGTCCG GTTTTCAACA AACCATGCAA ATGCTGAATG 3001 A3GGCATCGT TCCCACTGCG ATGCTGGTTG CCAACGATCA GATGGCGCTG GGCGCAATGC 3061 GCGCCATTAC CGAGTCCGGG CTGCGCGTTG GTGCGGACAT CTCGGTAGTG GGATACGACG 3121 ATACCGAAGA CAGCTCATGT TATATCCCGC CGTTAACCAC CATCAAACAG

GATTTTCGCC

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3181 TGCTGGGGCA AACCAGCGTG GACCGCTTGC TGCAACTCTC TCAGGGCCAG GCGGTGAAGG 3241 GCAATCAGCT GTTGCCCGTC TCACTGGTGA AAAGAAAAAC CACCCTGGCG CCCAATACGC 3301 AAACCGCCTC TCCCCGCGCG TTGGCCGATT CATTAATGCA GCTGGCACGA CAGGTTTCCC 3361 GACTGGAAAG CGGGCAGTGA GCGCAACGCA ATTAATGTAA GTTAGCTCAC TCATTAGGCA 3421 CCGGGATCTC GACCGATGCC CTTGAGAGCC TTCAACCCAG TCAGCTCCTT CCGGTGGGCG 3481 CGGGGCATGA CTATCGTCGC CGCACTTATG ACTGTCTTCT TTATCATGCA ACTCGTAGGA 3541 CAGGTGCCGG CAGCGCTCTG GGTCATTTTC GGCGAGGACC GCTTTCGCTG GAGCGCGACG 3501 ATGATCGGCC TGTCGCTTGC GGTATTCGGA ATCTTGCACG CCCTCGCTCA AGCCTTCGTC 3661 ACTGGTCCCG CCACCAAACG TTTCGGCGAG AAGCAGGCCA TTATCGCCGG CATGGCGGCC 3721 CCACGGGTGC GCATGATCGT GCTCCTGTCG TTGAGGACCC GGCTAGGCTG GCGGGGTTGC 3781 CTTACTGGTT AGCAGAATGA ATCACCGATA CGCGAGCGAA CGTGAAGCGA CTGCTGCTGC 3841 AAAACGTCTG CGACCTGAGC AACAACATGA ATGGTCTTCG GTTTCCGTGT TTCGTAAAGT 3901 CTGGAAACGC GGAAGTCAGC GCCCTGCACC ATTATGTTCC GGATCTGCAT CGCAGGATGC 3961 TGCTGGCTAC CCTGTGGAAC ACCTACATCT GTATTAACGA AGCGCTGGCA TTGACCCTGA 4021 GTGATTTTTC TCTGGTCCCG CCGCATCCAT ACCGCCAGTT GTTTACCCTC ACAACGTTCC 4081 AGTAACCGGG CATGTTCATC ATCAGTAACC CGTATCGTGA GCATCCTCTC TCGTTTCATC 4141 GGTATCATTA CCCCCATGAA CAGAAATCCC CCTTACACGG AGGCATCAGT GACCAAACAG 4201 GAAAAAACCG CCCTTAACAT GGCCCGCTTT ATCAGAAGCC AGACATTAAC

GCTTCTGGAG

PL 229 055 Β1

4261 AAACTCAACG AGCTGGACGC GGATGAACAG GCAGACATCT GTGAATCGCT TCACGACCAC 4321 GCTGATGAGC TTTACCGCAG CTGCCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA 4381 CACATGCAGC TCCCGGAGAC GGTCACAGCT TGICTG ^ AAG CGGATGCCGG GAGCAGACAA 4441 GCCCGTCAGG GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCGCAGCCAT GACCCAGTCA 4501 CGTAGCGATA GCGGAGTGTA TACTGGCTTA ACTATGCGGC ATCAGAGCAG ATTGTACTGA 4561 GAGTGCACCA TATATGCGGT GTGAAATACC GCACAGATGC GTAAGGAGAA AATACCGCAT 4621 CAGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG 4681 AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC 4741 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT 4801 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG 4861 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC 4921 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC 4981 TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT 5041 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT 5101 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC 5161 AGCCACT GGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA 5221 GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA 5281 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

CCACCGCTGG

PL 229 055 Β1

5341 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA 5401 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG 5461 GATTTTGGTC ATGAACAATA AAACTGTCTG CTTACATAAA CAGTAATACA AGGGGTGTTA 5521 TGAGCCATAT TCAACGGGAA ACGTCTTGCT CTAGGCCGCG attaaattcc AACATGGATG 5581 CTGATTTATA TGGGTATAAA TGGGCTCGCG ATAATGTCGG GCAATCAGGT GCGACAATCT 5641 ATCGATTGTA TGGGAAGCCC GATGCGCCAG AGTTGTTTCT GAAACATGGC AAAGGTAGCG 5701 TTGCCAATGA TGTTACAGAT GAGATGGTCA GACTAAACTG GCTGACGGAA TTTATGCCTC 5761 TTCCGACCAT CAAGCATTTT ATCCGTACTC CTGATGATGC ATGGTTACTC ACCACTGCGA 5821 TCCCCGGGAA AACAGCATTC CAGGTATTAG AAGAATATCC TGATTCAGGT GAAAATATTG 5381 TTGATGCGCT GGCAGTGTTC CTGCGCCGGT TGCATTCGAT TCCTGTTTGT AATTGTCCTT 5941 TTAACAGCGA TCGCGTATTT CGTCTCGCTC AGGCGCAATC ACGAATGAAT AACGGTTTGG 6001 TTGATGCGAG TGATTTTGAT GACGAGCGTA ATGGCTGGCC TGTTGAACAA GTCTGGAAAG 6061 AAATGCATAA ACTTTTGCCA TTCTCACCGG ATTCAGTCGT CACTCATGGT GATTTCTCAC 6121 TTGATAACCT TATTTTTGAC GAGGGGAAAT TAATAGGTTG TATTGATGTT GGACGAGTCG 6181 GAATCGCAGA CCGATACCAG GATCTTGCCA TCCTATGGAA CTGCCTCGGT GAGTTTTCTC 6241 CTTCATTACA GAAACGGCTT TTTCAAAAAT ATGGTATTGA TAATCCTGAT ATGAATAAAT 6301 TGCAGTTTCA TTTGATGCTC GATGAGTTTT TCTAAGAATT AATTCATGAG CGGATACATA 6361 TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC

CCGAAAAGTG

PL 229 055 Β1

6421 CCACCTGAAA TTGTAAACGT TAATATTTTG TTAAAATTCG CGTTAAATTT

TTGTTAAATC

6481 AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC

AAAAGAATAG

6541 ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT

AAAGAACGTG

6601 GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT

ACGTGAACCA

6661 TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA

6721 GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG

AAAGGAAGGG

6781 AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC

GCTGCGCGTA

6841 ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTCCCATTC GCCA

Sequence information:

LENGTH: 6,894 nucleotides

TYPE: nucleic acid

TOPOLOGY: plasmid

THREAD COUNT: one

TYPE OF PARTICLE: DNA

Claims (2)

1. The method of obtaining the recombinant Escherichia coli Rosetta (DE3) pLysS strain with the symbol Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH, characterized by obtaining a DNA fragment encoding the Deinococcus radiodurans trehalose synthase protein DSMZ 20539 of the formula 3 by amplification using oligonucleotide primers of formula 2 and symbols DraSTNdeF and DraSTXhoR, and the obtained DNA fragment is cloned into the pET30Ek / LIC plasmid vector, resulting in the plasmid vector of formula 4 and the symbol pET30Ek / LICDraSTH, containing the cloned DNA with the fragment encoding the trehalose protein synthase Deinococcus radiodurans DSMZ 20539, with fragments recognized by the restriction enzymes NdeI and XhoI, with the rbs ribosome binding site, with the kanamycin resistance gene, with the pBR322 plasmid origin, with origin f1, polyhistidine region, T7 promoter, S-Tag coding sequence, T7 transcription termination, with the lacI operator site then obtained the vector transforms into Escherichia coli Rosetta (DE3) pLysS cells. 2. Method for the production of Deinococcus radiodurans DSMZ 20539 trehalose synthase protein consisting in the production of the protein by cells of a recombinant bacterial strain in liquid media, characterized by the cultivation of cells of the Escherichia coli Rosetta (DE3) pLysS-pET30Ek / LICDraSTH bacterial strain at the initial temperature from 35 ° C to 38 ° C, and then, after obtaining the density of the cell suspension from 0.3 to 0.9, overexpression of the gene encoding the protein is carried out for a time ranging from 2 to 24 hours by adding an inducer, which is IPTG with a concentration of from 0.05 mM to 10 mM, and after separation of the cell suspension from the culture fluid, cell disintegration and initial thermal denaturation, the soluble proteins are isolated by centrifugation, and then they are purified using one purification step on coordinate-binding, reversible metal ions IDA-Co, then concentrated and suspended in a storage buffer, preferably with the following composition: 0.85% NaCl, 20% glycine erol (v / v), 5mM EDTA, 1mM DTT, 1mM PMSF, 0.05% IGEPAL. PL 229 055 Β1 Drawings Fig. 1. Amplified gene encoding Deinococcus radiodurans trehalose synthase protein DSMZ 20539 with additional recognition sites for NdeI and XhoI restriction enzymes, frame preservation, deleted stop codon - description.