Pettvdao2 recombinant plasmid providing synthesis of yeast trigonopsis variabilis d-amino acid oxydase (dao) in escherichia coli cells and recombinant escherichia coli strain c41(de3)/pettvdao2 as producer of dao

FIELD: biology, in particular gene engineering, microbiological industry, production of new beta-lactam antibiotics.

SUBSTANCE: constructed is recombinant plasmid DNA pETTvDAO2 providing high level of DNA fragment which encodes D-amino acid oxydase of Trigonopsis variabilis yeast in Escherichia coli cells. As a result of transformation of E.coli strain with disclosed recombinant plasmid and selection of transformed clones new recombinant Escherichia coli strain C41(DE3)/pETTvDAO2 as producer D-amino acid oxydase is obtained.

EFFECT: improved method for producing of cephalosporin antibiotics.

2 cl, 4 dwg, 2 tbl, 7 ex

 

The present invention relates to the field of biotechnology and, in particular, to genetic engineering and can be used in microbiological industry for preparation of semisynthetic cephalosporin antibiotics of the new generation. Available recombinant plasmid DNA pETTvDAO2 containing the DNA fragment encoding the oxidase D-amino acids of the yeast Trigonopsis variabilis, and providing a high level of expression of the enzyme in Escherichia coli cells, and the recombinant strain of E. coli C41(DE3)/pETTvDAO2 producing oxidase D-amino acids.

The most important intermediate product in the production of various antibiotics from the family of cephalosporin is 7-aminocephalosporanic acid (7-ACC) [1]. Conversion of cephalosporin C (Cefs) 7-ACC can be carried out either by chemical hydrolysis, requiring the use of extremely toxic compounds and special conditions of incubation at low temperatures, or by enzymatic transformations [2]. The most common two-stage method enzymatic transformation, involving the transformation of Cefs in glutaryl-7-ACC by the enzyme oxidase D-amino acids (DAO; EC 1.4.3.3), we get, for example, cells of microorganisms Aspergillus, Penicillium, Neurospora, Pseudomonas, Cephalosporium, Trigonopsis, and subsequent hydrolysis of glutaryl-7-ACC with the formation of 7-ACC under the action of a specific 7-beta-(4-for whom roxellanae) acylase (Gl7-ACA) from the cells of bacteria of the genus Comamonas, Pseudomonas or Arthrobacter [1, 3].

The increase in the production of semisynthetic beta-lactam antibiotics of the new generation (according to recent estimates [2] today the market of production of 7-ACC is about 20 billion US dollars per year) determines the increasing demand for the enzymes catalyzing the transformation of Cefs 7-ACC, including significant quantities of quality products oxidase D-amino acids and, consequently, stimulates interest in developing effective and efficient systems of production of this enzyme by methods of genetic engineering.

The level of technology

To date, installed sequence a number of natural genes encoding DAO constructed containing these sequences, the expression vectors and methods preparation of recombinant forms of the enzyme, in particular functionally active DAO Trigonopsis variabilis [4], Rhodotorula gracilis [5] in bacteria [6, 7] and yeast [8, 9].

The General disadvantages of the known methods heterologous expression of DAO in E.coli cells are low viability of recombinant strains producing and low yield of biomass even during prolonged cultivation on enriched environments, driven primarily by the toxicity of functionally active DAO to bacterial cells [10]. Toxic effect DAO cells most is the R recipient strains of E. coli is due to several factors: the overall metabolic stress of overproduction alien protein, depletion of the pool of D-amino acids necessary for the synthesis of the cell wall, releasing a by-product of the reaction DAO - hydrogen peroxide [5-7]. These factors greatly complicate the task of scaling the processes of obtaining DAO using recombinant strains of E.coli and provide the physiological stability of the product DAO, in the long fermentation and storage of bacterial strains producing DAO [10]. The solution to the problems associated with toxicity heterologic expressed DAO for bacterial host cells, additionally complicated by the fact that for most used in E.coli promoters characterized by a relatively high basal level of expression [11, 12, 13]. Thus, the task of creating a new stable and efficient E. coli strains - producers of DAO to successfully implement the scale fermentation processes, remains.

Disclosure of inventions

Taking into account peculiarities of expression of DAO in bacterial cells in solution of tasks of increasing output active recombinant DAO in E.coli is very promising is the development of new vector constructs containing the gene DAO, which would ensure the effective inducible enzyme synthesis at a low basal level of expression and the search for optimal from the point of view of stability and biomass yield of the system is the expression (a combination of "expressing vector/strain-recipient").

These aspects made the objective of the present invention.

This goal was achieved due to the fact that

1) in the collection of microorganisms was detected strain Trigonopsis variabilis, effectively synthesizing enzyme DAO (hereinafter TvDAO);

2) from this strain obtained gene TvDAO and DNA sequence that does not contain introns and coding of continuous full amino acid sequence of the enzyme;

3) constructed recombinant plasmid DNA pETTvDAO2 providing high and stable output TvDAO in Escherichia coli cells under conditions of induction;

4) the transformation expressing the plasmid pETTvDAO2 cells of the strain E. coli C43(DE3) were obtained recombinant Escherichia coli strain C43(DE3)/ pETTvDAO2 with a high level of induced synthesis and stable production of active DAO.

Strain Trigonopsis variabilis selected as the source to highlight the DAO gene, previously deposited in the all-Russian collection of microorganisms under the number VKM Y-2601.

The sequence of the gene TvDAO (SEQ ID No. 3), comprising the nucleotide sequence for the full amino acid sequence of the enzyme obtained by the method of polymerase chain reaction (PCR) using as the template the chromosomal DNA isolated from strain Trigonopsis variabilis VKM Y-2601, and as primers synthetic oligonucleotides from nucleotide sequence is mi SEQ ID No. 1 (primer tv1) and SEQ ID No. 2 (primer tv2), it is specific to its N - and C-terminal regions. Removal of the intron contained in the sequence obtained by PCR amplification with primers tv1/tv2, the primary PCR product was subjected to repeated PCR amplification with primers tv3 (SEQ ID No. 4) and tv5 (SEQ ID No. 5). The resulting re-amplification of the sequence SEQ ID No. 6, which does not contain intron, was used to construct expressing vector.

When creating recombinant plasmids for the expression of DAO was previously obtained intermediate vector pET-TetI (figure 2), containing as a genetic marker gene for resistance to tetracycline (TetR), which was replaced by the gene of beta-lactamase in the commercial plasmid RET. Use as a marker gene of resistance to tetracycline, and not traditionally used in such constructions resistance gene for ampicillin exempt from having to follow complicated purification of the target protein to remove impurities beta-lactamases. Expressing recombinant plasmid pETvDAO2 constructed by ligating the sequence SEQ ID No. 6 in the vector pET-TetI on the restriction sites NcoI and XhoI (figure 3).

To obtain a recombinant strain-producer TvDAO obtained plasmid pETvDAO2 was introduced into the cells of the strain of the recipient, which was elected the Escherichia coli strain C41(DE3)Selecting a strain of the recipient due to the fact, he and carries the gene for RNA polymerase of phage T7, which is necessary for efficient transcription of target genes in the vector constructs under the control of the promoter of T7 phage, b) defective in the synthesis of proteases, which significantly increases the yield of secreted heterologous proteins, and C) carrying a mutation that reduces the level of production T7 polymerase, which in turn reduces the basal level of gene expression from the T7 promoter [14]. The selection of productive and stable clones-producers among the tetracycline-resistant transformants of strain C41(DE3) proposed by plasmid was performed on the basis of production of functionally active DAO under cultivation in conventional selective media with the addition of the inducer isopropyl-D-thiogalactoside (IPTG) or lactose.

The obtained recombinant strain of E. coli C41(DE3)/pETTvDAO2 characterized by low basal level of expression of TvDAO and increased output functionally active oxidase under conditions of induction, which is on average about 1.5 times higher than in the known recombinant strains of Escherichia coli.

Thus, the present invention includes two objects.

The first object is a recombinant plasmid pETvDAO2 for synthesis oxidase D-amino acids Trigonopsis variabilis (TvDAO) in Escherichia coli cells, which consists of the coding TvDAO DNA fragment with a nucleotide placentas is of SEQ ID No. 6 and NcoI/XhoI fragment size 7,15 TPN the plasmid pET-Tet, obtained by replacing the gene of beta-lactamase in the vector RET gene for resistance to tetracycline (TetR) in the reverse orientation.

The second object of the invention is a recombinant Escherichia coli strain C41(DE3)/pETTvDAO2 producing oxidase D-amino acids.

A brief description of the figures.

Figure 1 - Comparison of the nucleotide sequences of the gene TvDAO Trigonopsis variabilis (Query, access number in Genbank Z80895) and the PCR fragment obtained on genomic DNA Trigonopsis variabilis VKM Y-2601 with primers tv1/tv2 (subject). The sequence of primer tv1.

Figure 2 - Physical and genetic map of the vector pET-TetD (a) and pET-TetI(B). The indicated position of indicator restriction enzymes cut sites, sites of promoter and terminator RNA polymerase of phage T7 (T7 prom and T7 term), the region of the beginning of replication (ColE1 origin, f1 origin), the gene of resistance to tetracycline (TetR), gene lac-repressor (lacI).

Figure 3 - Physical and genetic map of the plasmid pETTvDAO2. The position of the gene TvDAO (filled black), other symbols as in figure 2.

The implementation of the invention

When carrying out the invention, in addition to the methods disclosed in detail in the following examples used are well known in the art the techniques described in the manuals of molecular biology and genetic engineering [15, 16].

Example 1. Cultivation of strain Trigonopsis variabilis VKM Y-2601 and the determination of the activity of the oxidase.

Culture Trigonopsis vaiabilis grown in 5 ml YPD liquid medium, containing 1% yeast extract, 2% peptone, 2% glucose on a rocking chair at 28°With during the day. Cells are harvested by centrifugation, when the density of the culture reaches about 5 at 650 nm, washed in buffer (50 mm Tris pH8,0 5 mM EDTA), suspended in the same buffer (0.1 g wet cells per 200 ál of buffer) and frozen until use.

To determine the enzyme activity of DAO in the culture used colorimetric method [17], based on the detection of alpha-keto acid formed in the reaction. First get a rough extract, which frozen sediment cells destroy the vortex using glass beads (diameter 0.5 mm) with intermittent cooling on ice. Cellular debris is removed by centrifugation (13000 rpm, 10 min), 10-50 μl of the obtained clarified lysate incubated with 250 μl of 10 mm D-alanine in 50 mM phosphate buffer, pH 8.0, for 15 or 30 min, the reaction is stopped by adding 100 μl of a saturated solution of 2,4 dinitrophenylhydrazine in 2N HCl. After 5 min incubation at room temperature the solution was added 300 μl of 3M NaOH and 2.5 ml of distilled water and continue incubation for 10 min at room temperature, then measure the absorption of the solution at 550 nm, and comparing the result with a standard curve, constructed using pyruvic acid. Specific activity is expressed in units is armenta (mcmole substrate per minute at 25° C) per mg of total protein in the clarified extract. The total protein content determined by the Bradford method [15]. Thus, calculated specific activity of TvDAO in strain Trigonopsis variabilis VKM Y-2601 was 5 IU/mg protein.

Example 2. The allocation of genomic DNA Trigonopsis variabilis VKM Y-2601.

To the frozen cell suspension add 75 ál of 10% SDS to a final concentration of 2% and 200 μl acid washed glass beads (diameter 0.5 mm) and subjected to intense shaking on a vortex for 5 minutes. Then the suspension add 300 ál of saturated buffer THE phenol solution and shaken on a vortex for 30 sec. The mixture is centrifuged, the supernatant extracted twice with water saturated solution of chloroform. The aqueous phase is selected and add 5M potassium acetate solution (pH 6.0) to a final concentration of 1.5 M. the Solution is centrifuged, the supernatant add an equal volume of isopropanol. Precipitate the nucleic acids are collected by centrifugation, dissolved in 300 μl of 0.5 M potassium acetate, re-precipitated by adding 3 volumes of ethanol and dissolved in 100 μl of deionized water by heating for 30 min at 65°.

Example 3. PCR amplification of the gene sequence DAO Trigonopsis variabilis.

The primary structure of the gene DAO Trigonopsis variabilis and its mRNA is known (the access number in GenBank Z80895). This circumstance allows to design a "direct" and "reverse" PRA the measures for PCR amplification of the desired region of the gene. In this case, the primers used were that limit the sequence consisting of two exons and is located between intron:

5'-TGATAGGCAAATC CGTGCTC-3' straight-tv1-primer (SEQID No. 1) and

5'-GTAAACACGTCGCAGTCGTC-3' "reverse" tv2-primer(SEQID No. 2)

Genomic DNA Trigonopsis variabilis VKM Y-2601, isolated as described in example 2, are denatured by heating at 100°C for 5 minutes, placed on ice and subjected to 30 cycles of PCR in a 50 µl mixture of the following composition:

5 ál of 10x Taq-SE PCR buffer (SibEnzyme);

5 ál of genomic DNA (200 ng/ál);

5 μl of 3 μm primer tv1;

5 μl of 3 μm primer tv2;

5 μl of 2.5 mm dNTP each type;

25 µl of deionized water;

1 μl of Taq-SE polymerase (5 u/ál, SibEnzyme).

Conditions for PCR: 94°, 5' (denaturation), 94°, 30′′; 50°, 30′′; 72°,1' (amplification). After amplification, 5 µl of the PCR mixture is analyzed by electrophoresis in 1% agarose gel and identify homogeneous fragment size of about 1 TPN. Fragment isolated from the gel using the set Wizard PCR Preps Kit and subjected to automated sequencing using primers tv1, tv2. Based on the data sequencing was concluded accordance sequence selection expected (Fig 1)

Example 4. Obtaining and cloning of DNA sequences encoding the complete amino acid sequence DA Trigonopsis variabilis.

To obtain the DNA sequence encoding the complete amino acid sequence of TV-DAO, fragment, obtained by PCR amplification with primers tv1/tv2 (SEQ ID No. 3), remove the intron, which it is subjected to repeated PCR amplification with primers tv3/tv5 having the following structure:

NcoI

5'catgccATGGCTAAAATCGTTGTTATTGGGGCCGGTGTTGCCGGTTTAAC-3' tv5-primer (SEQ ID No. 4)

XhoI

5'CCCTCGAGCGGCCGCTAAAGGTTTGGACGAGTAAGA-3' tv3-primer (SEQ ID No. 5)

Used primers limit fragment of the DAO gene, including the beginning of the 2nd exon, located at position +125 sequence SEQID No. 3, and the termination codon TAG, located at position +1170 the same sequence, and add to this sequence initiation codon ATG broadcast and plot 8 encoding amino acids 1 exon. Primers are also sites for restricted NcoI and XhoI, missing in the coding sequence of the gene DAO. 1 ng of PCR fragment obtained as described in example 3 is subjected to 25 cycles of PCR amplification with primers tv5/tv3. From agarose gel isolated fragment size of about 1 TPN and is sequenced. The nucleotide sequence of this fragment is represented in SEQ ID No. 6.

Example 5. Construction of plasmids for the expression of TvDAO in E.coli.

Construction of recombinant plasmids was performed in several stages. As the base vector used PL is smido RET (Novagen).

A) Construction of plasmid pET TetD and pET TetI.

In order to remove AmpR gene from the plasmid pET21 and replace it with a resistance gene for tetracycline spent constructing the plasmid pET-Tet. To do this, first received a "box"that carries the gene for resistance to tetracycline: 2 μg of plasmid DNA pKRP12[18] restriction enzyme hydrolyzed in the > PST and was isolated from agarose gel, the fragment size of 1200 BP Plasmid RET hydrolyzed site > PST and ligated with the resulting "insert", using the following mixture: 1 μl of vector (20 ng/μl), 1 μl of fragment (50 ng/μl), 2 μl 5 × ligase buffer (Gibco-BRL), 5 μl water and 1 μl T4 DNA ligase (1 u/μl, SibEnzyme). The mixture is incubated for 14 hours at 12°, then warmed up for 15 min at 65°, cooled in ice and 5 μl of the mixture was used to transform competent cells of the strain E. coli JM 109 [15]. Selected tetracycline-resistant clones sensitive to ampicillin. Additional selection of the desired clones was performed using restrictive analysis of preparations of plasmid DNA (education fragments of size 1200, 1300, 4300 BP during the hydrolysis EcoRI+ > PST). The selected clones was further analyzed to determine the "orientation" insert token TetR through joint hydrolysis of restrictase ClaI+XhoI. The formation of fragments with a size of 1.7 TPN and 5.6 TPN indicates a "straight" orientation of the insertion marker TetR, and fragments the size of the m 2,7 TPN and 4, 6 TPN - about "reverse". The selected clones with the "direct" and "reverse" orientation marker TetR were identified as pET_TetD I. pET_TetI respectively, and used in further work. Physical and genetic map of the plasmid pET_TetD I. pET_TetI shown in figure 2.

B) preparation of recombinant plasmid pETTvDAO1 and pETTvDAO2.

Obtained in example 4, the fragment with SEQ ID No. 6 hydrolyzing restrictase NcoI and XhoI and are ligated with NcoI/XhoI fragment of the vector pET TetD size 7,15 KBP using the following conditions: 1 ál vector (20 ng/μl), 1 μl of fragment (50 ng/μl), 2 μl 5 × ligase buffer (Gibco-BRL), 5 μl water and 1 μl T4 DNA ligase (1 u/ál, SibEnzyme). The mixture was incubated overnight at 12°, then heated 15 min at 65° and cooled in ice. 5 μl of ligase mixture used to transform competent cells of the strain E. coli B21(DE3), and then on the selective medium containing tetracycline (at a concentration of 12 μg/ml), identify resistant to antibiotic transformants.

Selection of positive clones among the tetracycline-resistant transformants of strain E. coli BL21(DE3) have the ability to direct the synthesis of active DAO. To do this, individual clones were grown in 5 ml LB medium (0.5% of yeast extract and 0.5% NaCl, 1% tripton, pH of 7.2)containing 15 μg/ml tetracycline to the density OD ˜1,0, then add the inductor IPTG to a final concentration of 1 mm and continue stump the licensing overnight at a temperature of 23-25° C. Cells are harvested by centrifugation, suspended in 200 μl of THE buffer, add 100 ál of glass beads with a diameter of 0.1 mm and intensively shaken on a vortex. Receive the clarified lysates and determine the activity of the DAO, as described in example 1. Selected clones with specific activity of DAO in the range of 4-8 IU/mg protein. In the control (negative) samples activity no. One of the selected clones by sequencing contains insert with SEQ ID No. 6 in the desired orientation. Contained in this clone plasmid is designated as pETTv DAO1. To obtain the expression vector of TvDAO based plasmids pET_TetI NcoI/XhoI fragment size 1080 mon plasmids pETTv DAO1, the encoding gene TvDAO, perekodiruya in vector pET_TetI, and selected positive clone containing a plasmid with the full coding sequence of TvDAO (SEQ ID No. 6), which is designated as pETTv DAO2.

Example 6. Obtaining recombinant strain - producer TvDAO.

The obtained recombinant plasmid TvDAO1 and TvDAO2 transform the E. coli strains BL21(DE3) [F-, ompT, hsdSB(rB-, mB-), dcm, gal,DE3)], BL21(DE3)/plysS and C41(DE3). The strain E. coli BL21(DE3)/plysS contains additional plasmid with the gene of T7 phage lysozyme, which is an inhibitor of T7 polymerase. The presence of this plasmid provides additional control over the expression from the T7 promoter in the conditions of induction. Ø the AMM E. coli C41(DE3) is a derivative of BL21(DE3) and carries an additional mutation, reducing the level of transcription with T7 promoter, allowing to reduce the basal level of production and to improve the coupling between transcription and translation of target genes under the control of T7 promoter [12, 14].

Individual clones of the transformants are grown in conditions of low aeration for optimal product DAO, in 50 ml of LB medium, at a temperature of 30°With up to roughly 2.0 OE, then make the inductor IPTG to a final concentration of 1 mm and continue incubation, as described in [6]. At the end of cultivation determine the parameters of the culture growth and activity of the enzyme in bacterial cells. The results of these experiments are presented in table 1.

1,6
Table 1
Dynamics of accumulation of recombinant DAO under cultivation of various E. coli strains - producers
The growth of culture, OPUD. the DAO activity, units/mg proteinOutput DAO, IU/ml
Strains-producers DAOBefore inductionAfter inductionBefore inductionAfter induction
BL21(DE3)/pETTvDAO11,51,44105
BL21(DE3)/pETTvDAO21,85126
BL21(DE3)/pETTvDAO1/pLysS1,52,531510
BL21(DE3)/pETTvDAO2/pLysS1,73,521712
C41(DE3)/pETTvDAO11,6of 5.421920
C41(DE3)/pETTvDAO21,77,212325
DH5a(pCDAAO20 [6]1,0The concentration isThe concentration is10,6the 15.6

Based on these data it was concluded that the best results both in terms of biomass accumulation, and in terms of the output of the active enzyme network used as the recipient strain C41(DE3), and the vector plasmid pETTvDAO2. In addition, it was found that cells of strain C41(DE3), transformed expressing vector pETTvDAO2 are the most high stability during storage.

Accordingly, to obtain a recombinant strain-producer TvDAO was selected one of the clones obtained by transformation of strain E. coli C41(DE3) with plasmid pETTvDAO2.

Example 7. Characterization of the recombinant strain E. coli C41(DE3)/pETTvDAO2

Morphological features

Tile and have an elongated rod-like shape, if the division does not packouts.

Cultural characteristics

Cells grow well on commonly used nutrient media. Generation time is about 60 min in liquid LB-medium. 2-2,5% nutrient agar "Difco" formed round, smooth, yellowish colonies with smooth edges. When grown in liquid LB and YT environments formed smooth intense turbidity.

Physiological and biochemical characteristics

The optimal cultivation temperature from 25 to 30°C, the optimum pH of 7.6. Source of nitrogen are organic compounds (in the form of tryptone, yeast extract).

The yield of recombinant TvDAO (according to the results of determination of activity in samples of biomass producer strain was carried out based on a previously specified values of the specific activity TvDAO [6]) is about 150 mg/l when the titer of culture 1×109cells/ml

The comparison of obtained results with the data known from the literature, allows us to conclude that the level of production of recombinant TvDAO in the strain E. coli C41(DE3)/pETTvDAO2 on average 1.5 times higher than the productivity of known strains analogues [3-6].

Table 2
Dynamics of accumulation of recombinant DAO under cultivation of various E. coli strains - producers
The growth of culture, OPUD. the DAO activity, units/mg proteinOutput DAO, IU/ml
Strains-producers DAOBefore inductionAfter inductionBefore inductionAfter induction
BL21(DE3)/pETTvDAO11,51,44105
BL21(DE3)/pETTvDAO21,61,85126
BL21(DE3)/pETTvDAO1/pLysS1,52,531510
BL21(DE3)/pETTvDAO2/pLysS1,73,521712
C41(DE3)/pETTvDAO11,6of 5.421920
C41(DE3)/pETTvDAO21,77,212325
DH5a(pCDAAO20 [6]1,0The concentration isThe concentration is10,6the 15.6

The list of references.

1) Parmar A, Kumar H, Marwaha SS, Kennedy JF. 1998. Recent Trends in Enzymatic Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid (7-ACA). Critical Reviews in Biotechnology 18:1-12.

2) Barber MS, Giesecke U, Reichert A, Minas W. 2004. Industrial enzymatic production of cephalosporin-based beta-lactams. Adv. Biochem. Eng Biotechnol. 88:179-215.

3) Pollegioni L, Caldinelli L, Molla G, Sacchi S, Pilone MS. 2004. Catalyti properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources. Biotechnol. Prog. 20:467-73.

4) Lin L, Chien HR, Wang W, Hwang T, Fu H, Hsu W. 2000. Expression of Trigonopsis variabilis D-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants. Enzyme Microb. Technol. 27:482-91.

5) Molla G, Vegezzi C, Pilone MS, Pollegioni L. 1998. Overexpression in Escherichia coli of a recombinant chimeric Rhodotorula gracilis d-amino acid oxidase. Protein Expr. Purif. 14:289-94.

6) Alonso J, Barredo JL, Diez B, Mellado E, Salto F, Garcia JL, Cortes E. 1998. D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiology 144 (Pt 4):1095-101.

7) Garcia Lopez et AL. Enzymatic process for the preparation of cephalosporanic 7$B(G)-(4-Carboxybutanamide) acid by means of the modified enzyme D-aminoacid oxidase of trigonopsis variabilis produced in escherichia coli. U.S. patent 6,635,458, October 21, 2003.

8) Zheng H, Wang X, Chen J, Zhu K, Zhao Y, Yang Y, Yang S, Jiang W. 2005. Expression, purification, and immobilization of His-tagged D: -amino acid oxidase of Trigonopsis variabilis in Pichia pastoris. Appl. Environ. Biotechnol. 1-7.

9) Isoai A, Kimura H, Reichert A, Schorgendorfer K, Nikaido K, Tohda H, Giga-HamaY, Mutoh N, Kumagai H.2002. Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells. Biotechnol Bioeng. 80(1):22-32.

10) V.I. Tishkov, Boronenkov S.V. 2005. OXIDASE D: STRUCTURE, MECHANISM OF ACTION AND PRACTICAL APPLICATION. Biochemistry, 70:51-67.

11) Novagene Inc. Product catalogue. (1999), pET System Manual. 11thEdition.

12) Dumon-Seignovert L., Cariot G., and L. Vuillard (2004). The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expr. Purif. 37, 203-206.

13) Vethanayagam J, Flower A. 2005. Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase. Microbial Cell Factories 4:3.

14) Miroux B. and Walker, J.E. (1996). Over-production of proteins in Escherichia coli: mutant hosts that allow synhesis of some membrane proteins and globular proteins at high levels. J Mol. Biol. 260, 289-298.

15) Ausubel FM. 1988. Current protocols in molecular biology.

16) Sambrook J, Maniatis T, Fritsch EF. 1989. Molecular cloning a laboratory manual Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory.

17) Khang YH, Kim IW, Hah YR, Hwangbo JH, Kang KK.(2003) Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhancesactivity and stability of biocatalyst in the bioconversion process of cephalosporin C. Biotechnol Bioeng., 82(4), 480-8.

18) Reece, for K.S., and G.J. Phillips. 1995. New plasmids carrying antibiotic resistance cassettes. Gene 165:141-142.

1. Recombinant plasmid pETTvDAO2 for synthesis oxidase D-amino acids Trigonopsis variabilis (TvDAO) in Escherichia coli cells, which consists of the coding TvDAO DNA fragment with a nucleotide sequence of SEQ ID No. 6 and NcoI/XhoI fragment size 7,15 etc. n the plasmid pET-TetI, obtained by replacing the gene of beta-lactamase in the vector RET gene for resistance to tetracycline (TetR) in the reverse orientation.

2. Recombinant Escherichia coli strain C41(DE3)/pETTvDAO2 producing oxidase D-amino acids.



 

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FIELD: biotechnology.

SUBSTANCE: invention provides preparation containing microorganism strains Bacillus mycoides var "V.A.VNIISKhM No D138" and Azotobacter vinelandii var. "NP VNIISKhM No D24" with cell titer (0.8-1.2)·108 cl/mL taken at ratio (1.8-2.2):1, and further containing humates with space elements so that total composition is as follows: microorganism strains 25-30%, humates 8-12%, space elements 0.05-0.08%, and water the balance.

EFFECT: increased activity against various diseases and imparted growth stimulation effect.

5 ex

FIELD: biotechnology.

SUBSTANCE: invention provides preparation containing microorganism strains Bacillus mycoides var "V.A.VNIISKhM No D138" and Azotobacter vinelandii var. "NP VNIISKhM No D24" with cell titer (0.8-1.2)·108 cl/mL taken at ratio (1.8-2.2):1, and further containing humates with space elements so that total composition is as follows: microorganism strains 25-30%, humates 8-12%, space elements 0.05-0.08%, and water the balance.

EFFECT: increased activity against various diseases and imparted growth stimulation effect.

5 ex

FIELD: virology.

SUBSTANCE: invention proposes avian influenza virus strain, subtype H5N1. The strain is deposited in the Specialized virus collection of Virology center NII microbiology in Russian Federation defense Ministry at № 1141. Invention can be used in viral, serodiagnosis, immunological and molecular-biological methods of investigation.

EFFECT: valuable properties of strain.

1 tbl

FIELD: medicine, in particular drug screening for tuberculosis treatment.

SUBSTANCE: diagnosis is carried out by determination of tuberculosis mycobacteria sensitivity to isoniaside based on mutation detection in katG, inhA, oxyR/ahpC genes by conformational polymorphism of single-strain fragments and detection mutation in katA gene, which is responsible for abovementioned preparation resistance additionally in 10 % experiences. Also disclosed are primers for DNA of tested katA gene, amplification conditions and electrophoresis conditions. Nucleotide sequences of primer pair are disclosed in specification. Buffer containing in 30 mul Tris-HCl 10 mM, pH 8.8; KCl 50 mM; 0.5 % of Twin 20, formamide 5 %; MgCl 2.0 mM MgCl2; Tag-polymerase 1.5 U; each mucleoside triphosphate 200 muM; each oligonucleotide primer 0.1-0.15 muM; and sample 3 mul is used for amplification.

EFFECT: improved method for diagnosis of mycobacterium tuberculosis strain sensitivity to isoniaside.

FIELD: biotechnology; methods of purification of the oil polluted bleaching soil.

SUBSTANCE: the invention is pertaining to the biotechnology, in particular, to purification of the bleaching earth from pollution with the oil products. The method provides for introduction into the soil of the biomass of the consortium of petroleum oxidizing strains - Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3 and the biomasses of the strain of bacteria Azotobacter vinelandii IB 4 at the mass ratio of the biomasses equal to 1:1. The invention allows to activate the process of the oil products biodegradation, at that the degree of the degradation for 5 months compounds 81 %.

EFFECT: the invention ensures activation of the process of the oil products biodegradation with the degree of the degradation for 5 months making 81 percent.

5 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: disclosed is expression vector containing ColE1 replication system wherein homology of RNAI and RNAII of ColE1 replication initiating site with free tRNA is modified by one or more mutation in encoding region of RNAI or RNAII gene. Said mutations lead to substitution of one or more bases in loop 1, 2, 3 of RNAI or RNAII. Also described are bacterial cell transformed by said vector and method for production of desired protein from abovementioned bacterial cell. Claimed method includes cell culturing, protein isolation and purification thereof. Increased homology ratio of RNAI and RNAII with free tRNA causes increased amount of plasmida copies. Decreased homology ratio of RNAI and RNAII with free tRNA causes reduced metabolite load in cell that is highly for recombinant protein production.

EFFECT: improved method for controlling of plasmida copy amount.

12 cl, 1 dwg, 7 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to method for heterological expression of Neisseria meningitidis protein in E. coli. Expressed protein has immunogenic activity in relation to Neisseria meningitidis.

EFFECT: immunogenic proteins of high effectiveness.

9 cl, 26 dwg, 1 tbl, 23 ex

FIELD: gene and protein- engineering, medicine, pharmaceutical industry.

SUBSTANCE: invention relates to recombinant plasmide DNA pER-Gl is constructed, which provides synthesis of hybrid polypeptide containing human glucagone and intein in Escerichia coli cells. Producer of said hybrid polypeptide is obtained by transformation of E.coli ER2566 with pER-Gl plasmide. Method for production of human recombinant glucagone includes providing and cultivation of hybrid polypeptide producer containing human glucagone and intein followed by isolation and cleavage of said hybrid protein.

EFFECT: increased yield of human recombinant glucagone; simplified technology.

3 cl, 3 ex, 3 dwg

FIELD: biotechnology, genetic engineering, biochemistry, antibiotics.

SUBSTANCE: invention proposes a constructed recombinant plasmid pSVH0106 comprising the natural gene sequence for acylase of glutaryl-7-aminocephalosporinic acid of the strain Brevundimonas diminuta BKM B-1297 that encodes a full-scale enzyme precursor. As result of transformation of E. coli strain with the proposed recombinant plasmid and selection of transformed clones the novel strain of E. coli BL21(DE3)/pSVH0106 is obtained that represents a producer of G17ACA acylase providing high yield of active enzyme. Owing to the effective and stable expression of recombinant G17ACA acylase in the proposed system using the proposed invention allows scaling a process for preparing this enzyme used broadly in manufacturing antibiotics.

EFFECT: valuable properties of plasmid.

2 cl, 4 dwg, 2 tbl, 9 ex

FIELD: biotechnology, microbiology, molecular biology.

SUBSTANCE: invention relates to a method for the heterologous expression of proteins in Neisseria meningitides in E. coli cells. The protein to be expressed possesses the immunogenic activity with respect to the parent strain of Neisseria meniningitidis. The claimed invention provides the higher effectiveness in preparing immunogenic proteins.

EFFECT: improved method for preparing proteins.

6 cl, 40 dwg, 23 ex

FIELD: biotechnology, gene engineering, ecology.

SUBSTANCE: Escherichia coli cells containing plasmides with luxCDABE-genes are produced by recombinant DNA method under control of PkatG, PsoxS, PrecA, PgrpE inducible stress promoters. On the base of the same kit of Escherichia coli bacterial cells is developed for heptyl detection in environment medium.

EFFECT: accelerated method for heptyl detection.

1 tbl, 4 dwg

FIELD: biotechnology, gene engineering, medicine, pharmaceutical industry.

SUBSTANCE: plasmide pBSH2EGF DNA providing expression of human epidermal growth factor (hEGF) in E.coli strains, containing polymerase T7 gene, under controlling of t7 promoter, associated with seeding site of lac-repressor, is synthesized. Also disclosed is method for production of human epidermal growth factor including inducible expression thereof in cells of Escherichia coli strain BL21(DE3) transformed with plasmide pBSH2EGF DNA followed by isolation of target product from inclusion bodies.

EFFECT: effective agent for gene engineering, medicine, pharmaceutical industry, etc.

2 cl, 3 ex, 4 dwg

FIELD: molecular biology, biochemistry, medicine, oncology.

SUBSTANCE: invention relates to DNA sequences found in analysis of mDNA from squamous carcinoma cellular lines of different origin wherein these DNA sequences represent transcripts from rearranged genes SCCA1 and SCCA2. Result of rearrangement is formation of fused gene consisting of exon 2-7 of gene SCCA1 and exon 8 of gene SCCA2, or exons 2-7 of gene SCCA2 and exon 8 of gene SCCA1. Prepared expressing vectors comprising above said combinations of exons of two genes provide synthesis of corresponding fused protein in host-cell. Proposed sequences of nucleic acids and genetic constructions based on thereof represent novel agents for diagnosis squamous carcinomas.

EFFECT: valuable biological and medicinal properties of transcripts.

8 cl, 9 dwg, 1 tbl, 5 ex

FIELD: genetic engineering, biochemistry, virology.

SUBSTANCE: invention proposes recombinant plasmid pCI-neo-ODC-nsP1 and its variants pCI-neo-ODC-E2-L and pCI-neo-ODC-E2-M encoding proteins of Venezuelan equine encephalomyelitis (VEE) and ornithine decarboxylase protein. Plasmids are prepared by the sequential cloning gene-equivalent of non-structural protein nsP1 (fragments of L,M of the structural protein E2) of VEE virus as component of gene encoding enzyme ornithine decarboxylase being firstly into prokaryotic plasmid vector pET-23b and then into eucaryotic plasmid vector pCI-neo. Prepared plasmids can be used in the development of protective preparations used against Venezuelan equine encephalomyelitis virus as a prophylactic agent with respect of this pathogen.

EFFECT: valuable biological and medicinal properties of plasmid and agents.

3 cl, 2 tbl, 6 dwg

FIELD: microbiology, molecular biology, genetic engineering.

SUBSTANCE: invention relates to designing recombinant strains of E. coli carrying the cloned sequences of genome of meliodosis pathogen, B. pseudomallei, and determining different spectra of the medicinal resistance. Method involves transformation of E. coli JM107 competent cells with Kpn I-fragments of chromosomal DNA of the strain B. pseudomallei 56770 SMR2 ligated with Kpn I-restricts of vector pUC19 followed by selection of clones showing the combined resistance to antibacterial preparations of different classes. Then method involves carrying out the plasmid screening of the recombinant clones and hybridization analysis for detection of chromosomal DNA fragment by using B. pseudomallei chromosome sequences as a probe. Then method involves assay of the presence of the expression product of chromosomal DNA cloned sequence by immunoblotting method with immunoglobulins of specific meliodosis antsera. The prepared recombinant strain ZV1 is deposited in the State Collection of pathogenic microorganisms "Mikrob" at number KM167 and it shows resistance to pefloxacin and streptomycin. Use of the invention provides carrying out investigations of molecular-genetic bases of the multiple medicinal resistance of B. pseudomallei and to study the functional role of separate proteins in formation of the polyresistance in the meliodosis pathogen.

EFFECT: valuable properties of strain.

1 dwg, 1 tbl, 2 ex

FIELD: biotechnology, gene engineering, medicine.

SUBSTANCE: new chimerical enzyme proteins, in particular proteins comprising fragments of porcine uricase and baboon uricase (PBC and PKS) are obtained by increasing of number of accessible PEG-binding sites in mammalian uricase by substitution of at least one fragment of mammalian lysine-free native enzyme amino acid sequence with homological lysine-containing uricase fragment of mammalian belonging to another specie and screening of uricase variants based on activity and immunogenicity after conjunction with PEG. Disclosed uricase variants maintain after PEGylation sufficiently the same uricolytic activity that non-modified enzyme and are sufficiently non-immunogenic ones. Also describes are methods for production of new chimerical uricase proteins by recombinant DNA technologies and vector constructs and expression systems used therefore.

EFFECT: intermediates for medicines with low immunogenicity and enhanced bioavalibility.

16 cl, 14 dwg, 4 tbl, 7 ex

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