Recombinant plasmid psvh0106 providing synthesis of gl7aca acylase in escherichia coli cells, recombinant strain of escherichia coli bl21(de3)/psvh0106 as producer of gl7aca acylase
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
The present invention relates to the field of biotechnology, in particular genetic engineering, and can be used in microbiological industry for preparation of semisynthetic beta-lactam antibiotics of the new generation.
Features recombinant plasmid pSVH0106 containing a DNA fragment which encodes the complete amino acid sequence of the acylase glutaryl-7-aminocephalosporanic acid strain Brevundimonas diminuta BKM-1297, and providing a high level expression in Escherichia coli cells, and the recombinant strain E. coli BL21(DE3)/pSVH0106 - producer Gl7-acylase
The level of technology
The most important connection-the predecessor to get antibiotics from a family of cephalosporin is 7-aminocephalosporanic acid (7-ACA) . Conversion of cephalosporin C in 7-ACA 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 transformation. Known processes of enzymatic transformation can be divided into 2 groups: a) one-step using, for example, the cephalosporin C-acylase from Pseudomonas and b)two-stage, including the conversion of cephalosporin C glutaryl-7-ACA (Gl7ACA) using the enzyme oxidase D-amino acids (DAO; EC-22.214.171.124), what particular derived from Trigonopsis variabilis Aspergillus, Penicillium, Neurospora, Pseudomonas, Cephalosporium, and subsequent hydrolysis of glutaryl-7-ACA with the formation of 7-ACA or under the action of a specific 7-beta-(4-carboxymethylamino) acylase (Gl7-acylase) , or under the action of enzymes of other classes related to gamma-glutamyl-tranferases or cephalosporin-C-deacetylases .
The most practical interest for biocatalytic synthesis of 7-ACA is the enzyme Cl7-acylase , related to the extensive family of penicillin-amidase (EC 126.96.36.199), capable of converting the penicillins and cephalosporins in valuable intermediate compounds used for the production of new antibiotics [6,7]. Gl7-acylase are heterotetramer consisting of 2 α and 2 β abietinic  and is characterized by high activity in relation to Gl7ACA at low activity against cephalosporin C.
To date, the known primary structure of bacterial genes Gl7-acilis from various strains of Pseudomonas described recombinant plasmid DNA for synthesis of these enzymes in the cells of E. coli and Bacillus subtilis, as well as the methods of production of recombinant forms of the enzymes with their use [4, 8-14].
The General disadvantages of the known methods heterologous expression Gl7ACA-Atzilut are the low level of synthesis of the recombinant product  and is largely associated with et is m, the complexity of processes scale fermentation strains [16,17], as well as the necessity to release derived drugs Gl7-acilis from impurities nonspecific beta-lactamases . In this regard, it is important to develop new means of providing higher output Gl7-acylase upon receipt by recombinant DNA technology and the simplification of the methods of purification of these enzymes.
Disclosure of inventions
The solution to increasing output active recombinant Gl7-acylase involves the identification of bacterial strains, effectively synthesizing in natural conditions this enzyme, the allocation of them encoding the enzyme genes and the creation of optimal vector designs for their efficient and stable expression in heterologous bacterial cell. These aspects made the objective of the present invention.
This goal was achieved due to the fact that
1) in the collection of microorganisms detected strain (Brevundimonas diminuta BKM-1297), actively producing Gl7-acylase (BrdGl7ACA);
2) from this strain obtained gene Gl7-acylase and determined the DNA sequence encoding the complete amino acid sequence of the enzyme;
3) constructed recombinant plasmid DNA pSVH0106 for synthesis BrdGl7ACA in cells of Escherichia coli with high yield;
4) in the transformation of cells expressing the plasmid pSVH0106 receiving the recombinant Escherichia coli strain BL21(DE3)/ pSVH0106 with a high level of inducible synthesis of active Gl7-acylase.
Strain Brevundimonas diminuta VKM B-1297 previously deposited in the all-Russian collection of microorganisms and selected us as the most active producer of Gl7ACA-acylase (BrdGl7ACA).
Complete coding sequence of a gene BrdGl7ACA obtained by polymerase chain reaction (PCR) using as the template the chromosomal DNA isolated from strain Brevundimonas diminuta VKM B-1297, and as primers synthetic oligonucleotides from nucleotide sequences SEQ ID No. 12 (primer 7-aca-F) and SEQ ID No. 13 (primer 7-aca-R), it is specific to the N - and C-terminal regions of the coding parts (from position 103 to position 2265) isolated gene fragment BrdGL7ACA with SEQ ID N06.
This sequence encodes a protein size KD (SEQ ID N0 7), possessing a high degree of homology with other known precursors of bacterial Gl7ACA-Atzilut, and includes N-terminal signal sequence for secretion and a sequence encoding two subunits of the enzyme, separated by spacer elements peptide.
At the first stage of creating a recombinant plasmid for expression of the DNA fragment encoding Gl7ACA-acylase, was designed vector-media Rast, physical and genetic map of which is presented in figure 3. This vector includes a promoter and a terminator RNA polymerase of phage T7, divided by the area of polylinker, the p15A replicon and knR gene, encoding aminoglycoside-3-phosphotransferase and ensure the sustainability of E.coli cells carrying a plasmid to kanamycin. Use as a marker gene of resistance to kanamycin, and not traditionally used in such constructions the gene of resistance to ampicillin, free from the need for further complex purification of the target protein to remove impurities beta-lactamases.
Expressing recombinant plasmid pSHV0106 obtained by embedding the coding sequence of the gene BrdGl7ACA in vector rst the restriction sites EcoRI and Sac I (figure 4).
Recombinant strain producing BrdGL7ACA was obtained by transformation of Escherichia coli cells BL21(DE3) constructed a plasmid pSVH0106. The choice of the strain of the recipient due to the fact that he 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, and the fact that this strain is defective in the synthesis of proteases, which significantly increases the yield of secreted heterologous proteins. Synthesis BrdGL7ACA in strain BL21(DE3)/ pSVH0106 is under cultivation in conventional selective media with the addition of the inducer isopropyl-D-thiogalactoside (IPTG) or lactose.
The obtained recombinant Escherichia coli strain BL21(DE3)/ pSVH0106 characterized physiological the stability (decreased activity of enzyme produced after 50 generations does not exceed 20%) and high yield of active product, which is not less than 1.5 times the productivity of known strains.
Thus, the present invention includes two objects.
The first object is a recombinant plasmid pSVH0106 for synthesis of the acylase glutaryl-7-aminocephalosporanic acid (Gl7ACA-acylase in Escherichia coli cells, which is characterized in that it contains a fragment of DNA with the nucleotide sequence SEQ ID No. 6 from nucleotide at position 103 to the nucleotide in position 2265 encoding the complete amino acid sequence Gl7ACA-acylase strain Brevundimonas diminuta VKM B-1297, built-in restriction sites EcoRI and SacI in the area polylinker vector rest consisting of a fragment of the modified area polylinker plasmids 21d containing the promoter and terminator of RNA polymerase of phage T7, divided area polylinker, and fragment of plasmid rsus containing the replicon RA and the gene for resistance to kanamycin, connected together, as shown in Fig 3.
The second object of the invention is a recombinant Escherichia coli strain BL21(DE3)/ pSVH0106-producer BrdGL7ACA.
Technical result achieved in the implementation of the present invention is the creation of new tools (expression vector and the recombinant strain)enhancing the quantity (yield) and quality (purity beta-lactamase) received recom is Yantai Gl7ACA-acylase.
Features of the present invention are explained the best options for its implementation with reference to figures.
A brief description of the figures.
Figure 1 Comparison of the nucleotide sequences of the Central coding portion of the gene Gl7ACA strain Brevundimonas diminuta VKM-1267 (Query) and the homologous gene of glutaryl acylase of Pseudomonas SY-77 (Sbjct) using the program BLAST. The degree of similarity of the sequences is 98%.
Figa and 2B Compare the amino acid sequences of known Gl7ACA Atzilut from Pseudomonas sp.130 (AAC34685), Pseudomonas sp.SY-77 (AAN39264), Pseudomonas sp.THA1 (AAP68796) c sequence BrdGl7ACA. The degree of similarity of the sequences is 98%.
3 Physical and genetic map of the vector pACT7. The indicated position of indicator restriction enzymes cut sites, sites of promoter and terminator RNA polymerase of phage T7 (Tprom and T7 term), the starting area replication (p15Aori), the gene for resistance to kanamycin (denoted by KN(R), filling black).
4 Physical and genetic map of the plasmid pSVH0106. The position of the gene BrdGl7ACA, (filled gray), and the remaining notation is as in figure 3.
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 [18, 19].
Example 1. Identification of strain Brevundimonas with a high level of biosynthesis Gl7ACA-acylase
Culture isolates of different strains of laboratory collection were grown in Erlenmeyer flasks with a volume of 100 ml in 15 ml of medium containing 2% casein hydrolysate, 0.5% sodium glutamate, 0.5% of yeast extract, 0.2% of corn extract, 0.1% of glutaric acid (pH 8,2) at 28°and With constant stirring (150 rpm) for 16 hours. When the density of the culture reached a value of 2 OE at a wavelength of 600 nm, the cells were collected by centrifugation (6000 rpm; 10 min; +4°C), washed in buffer (50 mm Tris pH 8.0 50 mM EDTA), suspended in the same buffer (0.1 g wet cells per 200 ál of buffer) and stored until use in frozen form.
In each of the samples was determined glutaryl-atillasoy activity, for which we used the colorimetric method similar to the method proposed for the determination of 6-aminopenicillanic acid . For this purpose, 100 μl of 65 mM solution glutaryl-7-ACA in 0.1M phosphate buffer (pH 7.0) is added to 900 μl of the cell suspension prepared in the same (0.1M phosphate) buffer containing 3 mg/ml of clavulanate potassium, and incubated the mixture at 37°from 30 minutes to 4 hours. After a certain period of time the mixture is centrifuged, selected 500 μl of the supernatant and mix it with 3 ml of a mixture (2:1) 20% acetic acid and 0.5 M NaOH to stop the reactions is. The sample add 0.5 ml of p-dimethylaminobenzaldehyde (PDAB) and after color development measure the absorbance at 415 nm. One unit of enzyme activity is accepted amount required to convert 1 μmol of substrate in these conditions for 1 min incubation.
Certain level so acylase activity in 64 analyzed strains ranged from 0,ed/l to about 4 IU/l culture. The highest activity possessed strains Brevundiminas diminuta VKM B-1297 and Brevundimonas vesicularis VKM B-974-derived from the all-Russian collection of microorganisms. Strain Brevundimonas diminuta VKM B-1297 was selected for further work.
Example 2. Obtaining chromosomal DNA from strain Brevundimonas diminuta VKM B-1297
To 200 μl of cell suspension of strain B.diminuta VKM B-1297, obtained as described in example 1, add 10% SDS to a final concentration of 2% and 20 ál of proteinase K (3000 u /ml) and incubated first for 30 min at 37°and then 15 min at 75°C. To the suspension is added 200 μl acid washed glass beads (diameter 0.1 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 is taken and extracted twice with water-saturated solution of chloroform. The aqueous phase is selected and add 5M RA is solution of potassium acetate (pH 6.0) to a final concentration of 1.5 M. The solution is centrifuged and discard the precipitate of the potassium salt of SDS. To 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°. Obtained by this method, chromosomal DNA of strain Brevundiminas diminuta VKM B-1297 used for obtaining the gene encoding Gl7ACA - acylase.
Example 3. The selection of the Central fragment of the gene BrdGl7ACA
Previously, the comparison of amino acid and nucleotide sequences of bacterial Gl7ACA-acilis using the information available through the Internet on the website of the National Center for Biotechnology Information, USA. Identified the most conservative areas GL7ACA-acilis of bacterial origin and synthesized two synthetic oligonucleotide brd1 (SEQID N01) and brd2 (SEQ ID N02), bounding the Central coding sequence of a gene Gl7ACA-acylase:
For the implementation of PCR of chromosomal DNA Brevundiminas diminuta VKM B-1297, selected as described above, generatorerror by heating at 100°C for 5 minutes, placed in ice, subjected to 30 cycles in a 50 µl mixture of the following composition:
5 ál of 10x Taq-SE PCR buffer (SibEnzyme)
5 µl of genomic DN is (200 ng/µl)
5 μl of 3 μm primer brd1
5 μl of 3 μm primer brd2
5 μl of 2.5 mm dNTP (a mixture of all four types of deoxynucleotidase)
25 µl of deionized water
1 μl of Taq-SE polymerase (5 u/ál, Sibenik)
The conditions of the reaction: 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. When the separation in electrophoresis identified homogeneous fragment size of about 1 TPN. The fragment was isolated from gel using the set Wizard PCR Preps Kit (Promega, USA) in accordance with the manufacturer's instructions) and subjected to automated sequencing on the device ABIPrizm 3100 DNA Sequencer using primers brd1, brd2 and set Applera "fluorescent Big dye Cycle sequencing kit".
The comparison of the obtained nucleotide sequences from the GenBank database using the BLAST program showed that it possesses a high degree of homology with the corresponding coding sequences of other bacterial Gl7AC-allas (Fig 1). On this basis it was concluded that this fragment corresponds to the Central part of the gene Brd Gl7ACA - acylase strain Brevundiminas diminuta VKM B-1297.
Example 4. Receiving, sequencing and analysis of DNA fragment comprising the complete coding sequence of a gene BrdGl7ACA
The DNA fragment comprising the complete coding sequence, which are square using PCR amplification with primers (BrdGl7ACA_F with SEQID N03 and BrdGl7ACA_R with SEQ ID N04), specific to the conservative sites flanking the full coding sequences of known genes Gl7ACA-acilis own right.
1 µg of genomic DNA is subjected to 25 cycles of PCR amplification with primers BrdGl7ACA_F / BrdGl7ACA_R and a set of Expand Long Template PCR system (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions.
The reaction mixture was separated by electrophoresis in 1% agarose gel and detect the reaction product is a piece of approximately 2, 25 TBN Fragment isolated from the gel using a set of "Wizard PCR preps Purification System (Promega, USA) and is sequenced using primers BrdGl7ACA_F, BrdGl7ACA_R, brd1, brd2 and brd 3 (SEQ ID N05).
So established the nucleotide sequence of the gene BrdGl7ACA given in the list under the number SEQ ID N06. Derived from her amino acid sequence of the protein BrdGl7ACA (SEQ ID N07) has a 98%similarity with the amino acid sequences of other known bacterial GL7ACA - allas (Fig 2), with differences that are localized in sites remote from the sites of the active centre of the enzyme and areas misbehaving contacts . These data provide a high degree of probability to conclude that the enzyme BrdGl7ACA consistent with other bacterial Gl7ACA-ullasam and their enzymatic and physico-chemical properties.
Example 5. Design vector-media pACT7<> Vectors based on the promoter of T7 phage provide high expression of heterologous genes in E. coli strains synthesizing T7 polymerase , and commercially available . At the same time, it is known that immobilized preparations Gl7ACA-acilis used for biotransformation of cephalosporin antibiotics, should not contain impurities of beta-lactamases. The presence of beta-lactamase also complicates quantitative analysis of the levels of production Gl7ACA-acilis in recombinant strains-producers. Therefore, for the expression of BrdGl7ACA and other biotransformation enzymes antibiotics, free from impurities of beta-lactamases, it is advisable to use vectors carrying the marker plasmid stability, non-AmpR. Such vectors also possess a higher segregation stability , which can be further enhanced through the use of the p15A replicon instead of ColE1 replicon.
Design vector pACT7 that meet the specified conditions, were carried out in several stages.
A) Construction of intermediate plasmids pET2ldRI.
C using the method of "inverse PCR and primers pETR1_F (SEQ ID N08) and pETR1_R (SEQ ID N09) on the matrix plaidy pET21d  using a set of Expand Long Template PCR system have a unique PCR fragment size of 5.4 TPN, which is isolated from the agarose gel as described in example 4. 100 ng of the resulting fragments is enta hydrolyzing 5 units restrictase EcoRI (Fermentas) and are ligated using T4 DNA ligase. Received ligase mixture transform competent cells of Escherichia coli strain XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacIqZM15 Tn10 (Tetr)] (Stratagene, USA), then from the obtained ampicillin-resistant transformants isolated preparations of plasmid DNA using the set Wizard MiniPreps Kit (Promega, USA). Obtained DNA samples analyze joint hydrolysis of restrictase EcoRI/ > PST and BamHI/ > PST and select "positive" clones containing EcoRI/ > PST fragment sizes 4100 1300 and P.N. however due to the deletion of the BamHI site in BamHI/ > PST hydrolysate DNA plasmid clones present a unique fragment 5400 mo, while in the drug pET21d plasmid DNA, hydrolyzed BamHI/ > PST, found fragments of size 1300 and 4100 P.N. Several positive clones was checked by sequencing using primers Trga (SEQ ID N0 10) and T7term (SEQ ID N0 11) and select clone with modified so the plot of polylinker, not containing nonspecific mutations that indicate how pET21dR1.
B) Construction of plasmids pET21dRl/Tet.
To remove the AmpR gene from plasmid pET21dR1 and replace it with a resistance gene for tetracycline spent constructing plasmids pET21dR1/Tet. To obtain the "insert" carrying the gene of resistance to tetracycline 2 μg of plasmid DNA pKRP12 restriction enzyme hydrolyzed in the > PST and allocated and the agarose gel, the fragment size of 1200 BP As vector used gidralizovanny site > PST plasmid pET21dR1. Used the following criteria ligation: 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 is incubated for 14 hours at 12°C, then warmed up for 15 min at 65°C, cooled in ice and 5 μl of the mixture was used to transform competent cells of E. coli strain JM109. 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). One of the selected clones identified as pET21dR1/Tet and used in further work.
C) Construction of plasmids pACYCpET.
To obtain the derived vector pET21dRI with the p15A replicon DNA plasmid pACYC177  hydrolyzed together with restrictase BamHI+ > PST and was isolated from agarose gel, the fragment size 3000 BP, including the region of the replicon RA, the gene for resistance to kanamycin and C-terminal coding part of the gene beta-lactamase (resistance to ampicillin). Plasmid pET21dR1 hydrolyzed together BglII+ > PST and was isolated from an agarose gel slice 1.5 TPN, including the area of polylinker, the promoter and terminator T7-phage and N is Onaway the coding part of the gene beta-lactamase. Two fragments ligated received ligase mixture was used to transform E. Coli strain Xl1Blue and selected transformants resistant to both ampicillin and kanamycin.
Isolated from cultures of the obtained clones plasmid DNA was analyzed by restriction analysis with the endonucleases SmaI and selected clones, forming fragments with a size of 3.2 and 1.2 TPN. One of the selected clones identified as pACYCpET.
G) Construction of vector pACT7.
Removal of the gene beta-lactamase from the vector pACYCpET preparation of plasmid DNA hydrolyzing jointly by restrictase NaeI/FspI, isolated from the agarose gel, the fragment size 3,4 TPN, are ligated "himself" blunt ends" and ligase mixture transform competent cells of E. coli strain JM109. Selected kanamycin-resistant and ampicillin-sensitive transformants produce plasmid DNA and identify the correct clone according to restrictive analysis (the presence of fragments of size 1.2 and 2.2 TPN in SmaI-hydrolysates preparations of plasmid DNA).
Example 6. Construction of plasmids for expression BrdGL7ACA in E.coli.
First PCR obtain a DNA fragment of strain Brevundiminas diminuta VKM B-1297, containing the full coding sequence BrdGl7ACA (no not adjacent coding sequences) and flanked by unique restriction sites.
To do this, 1 μg of genomic DNA of strain VKM B-1297 subjected tikla PCR amplification with primers 7-aca_F/7aca_R (SEQ ID N012,13) with a set of Expand Long Template PCR system (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Used primers restrict gene fragment BrdGL7ACA, including initiation codon broadcast, located at position (+103) sequence SEQID N06, and termination codon TGA, located at position+2265 the same sequence and are sites for restricted EcoRI and SacI, missing in the coding sequence of the gene BrdGl7.
The reaction mixture was separated by electrophoresis in 1% agarose gel and detect the reaction product is a piece of a size of about 2.16 TPN isolated from an agarose gel slice hydrolyzing restrictase EcoRI/SacI and clone into the EcoRI/SacI vector pET21dR1/Tet (see example 5 B). Select the tetracycline-resistant transformants of strain E. coli JM109 according to the criterion of formation of fragments with a size of 2.2 and 6.5 TPN in EcoRI/SacI hydrolysates obtained preparations of plasmid DNA.
In order to identify clones carrying an insert with the intact gene BrdGL7ACA possible without mutations introduced through PCR amplification, check the ability of the selected plasmid to direct the synthesis of functionally active BrdGL7ACA when they transfer into the E.coli strain BL21(DE3) .
For this individual transformants of the strain E. coli BL21(DE3)carrying the resulting plasmid, grown in LB-medium containing 10 μg/ml tetracycline in a volume of 5 ml to OP˜ and 1.0. After this culture make IPTG to ejnoy a concentration of 1 μm for the induction of the promoter of phage T7 and continue culturing for 18 hours at a temperature of 25-30° C. Cells are harvested by centrifugation and carry out the determination of the activity of Gl7ACA-acylase, as described in example 1. Selected positive clones is sequenced using primers T7prom, T7term, brd1, brd2, brd3 and identify the clone carrying the box with the intact gene BrdGl7ACA, the coding sequence of which is identical to the sequence of the DNA fragment from position+103 to position+2265 specific coding sequence of a gene BrdGl7ACA shown in the sequence listing under the number SEQ ID N07. The plasmids isolated from selected clones represent pSVH18.
To get expressing BrdGl7ACA plasmid c-p15A replicon EcoR1/Sac1 fragment from the genome BrdGl7ACA of plasmids pSVH18 clone in the vector rest with the formation of plasmid pSVH0106.
Example 7 preparation of recombinant strains of E. coli using plasmid pSVH18 and pSVH0106.
To obtain strains producing recombinant BrdGl7ACA using standard methods  carried out the transformation of the recipient strain E. coli BL21(DE3)  obtained recombinant plasmid DNA. On plates with LB medium containing 10 mg/l tetracycline, taken individually tetracycline-resistant transformants obtained using plasmid DNA pSVH18. Primary transformants were cultured in LB liquid medium containing 10 mg/l tetracycline, conduct the induction of synthesis BrdGl7ACA using the-W IPTG, as described in example 6. One of the selected clones was designated as E. coli BL21(DE3)/pSVH18, it is cultivated in 5 ml liquid LB medium containing 10 mg/l tetracycline to OP˜ 5,0 add to the culture an equal volume of sterile 30% glycerol, poured into 500 ál in sterile 2 ml tubes, frozen and stored at - 70°C.
Similarly receive primary transformants of the strain E. coli BL21(DE3) with plasmid pSVH0106. Individual kanamycin-resistant transformants grown in LB liquid medium with the addition of 30 mg/l kanamycin, check on the ability to synthesis of functionally active BrdGl7ACA, select single clone of E. coli BL21(DE3)/ pSVH0106 and stored at - 70°C.
Example 8 determination of the levels of production of recombinant BrdGl7ACA and physiological stability of the producing ability of recombinant strains.
For comparative quantitative determination of the expression level BrdGl7ACA in the resulting recombinant strains of E.coli BL21(DE3)/ pSVH18 and E.coli BL21(DE3)/ pSVH0106 cells grown in Erlenmeyer flasks with a volume of 300 ml in 50 ml LB medium with the addition of the appropriate antibiotic at a temperature of 25°to a600of 1.0 OE. Next make the inductor - IPTG to concentrations of 0,2mM and spend incubation for 22 hours. Periodically selected aliquots of the cell suspension and use them to determine crop growth and activity of the enzyme as described in example 1./p>
As can be seen from the presented data (table 1), the enzyme activity reaches its maximum after 20 hours of incubation; however, in the strain E. coli BL21(DE3)/ pSVH0106 it is significantly higher (the maximum value is 1200 IU/ml culture)than in E.coli strain BL21(DE3)/ pSVH18.
Accumulation dynamics BrdGl7ACA the cultivation of recombinant strains of E.coli BL21(DE3)/pSVH18 and E.coli BL21(DE3)/pSVH0106
|Time of cultivation after induction (h)||Activity, IU/ml culture||The density of the culture, And600nm|
Checking physiological stability producing activity of recombinant strains in conditions of continuous cultivation is as follows.
1. Spend the first cycle of cultivation in LB medium as described above, and at the end of cultivation determine the activity of the acylase (1st cycle of cultivation, corresponding to about 30 generations).
2. 1 ml of culture, polucen the th after the first cycle, used as inoculum, which contribute in 50 ml of fresh LB medium, and repeat the whole cycle Yeshe times (2nd cycle of cultivation, about 5 generations).
3. Repeat the procedure 4 more times - up to 50 generations.
As shown in the Table 2 data, strain BL21(DE3)/ pSVH0106 has a higher physiological stability in comparison with the strain BL21(DE3)/ pSVH18.
The physiological stability of the products BrdGl7ACA in the cells of recombinant E. coli strains BL21(DE3)/pSVH18 and E.coli BL21(DE3)/pSVH0106
|Strain||Activity BrdGl7ACA (IU/ml culture) depending on the number of generations|
|E. coli BL21(DE3)/pSVH18||800±15||750±12||700±20||650±15||600±20|
|E. coli BL21(DE3)/pSVH0106||1200±30||1150±25||1120±20||1100±20||1070±15|
Example 9. Characterization of the recombinant strain E. coli BL21(DE3)/pSVH0106
Considering the fact that the specific activity allas homologous BrdGL7ACA, the average is about 10,000 IU/mg protein, the yield of recombinant BrdGL7ACA in strain BL21(DE3)/pSVH0106 equal primer is 100 mg of protein per liter of culture producer. The comparison of the received data with previously known literary information about the characteristics of recombinant E. coli strains producing bacterial Gl7ACA_, suggest that the level of production of recombinant Gl7ACA_ in strain BL21(DE3)/ pSVH0106 not less than 1.5 times the productivity of known strains [9,12].
Cells have an elongated rod-like shape, when the division is not packouts.
Cells grow well on commonly used nutrient media. The generation time of about 30 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 level of synthesis BrdGL7ACA (according to the definition of activity in the samples of biomass producer strain) is about 100 mg/l under the title culture 1×109cells/ml
1. Recombinant plasmid pSVH0106 for synthesis of the acylase glutaryl-7-aminocephalosporanic acid (Gl7-acylase in Escherichia coli cells, which is characterized in that it contains a fragment of DNA with the nucleotide sequence SEQ ID No. 6 from nucleotide at position 103 to the nucleotide in position 2265 encoding the complete amino acid sequence Gl7-acylase strain Brevundimonas diminuta BKM-1297, built-in restriction sites EcoRI and SacI in the area polylinker vector rest consisting of a fragment of the modified area polylinker plasmid pET21d containing the promoter and terminator of RNA polymerase of phage T7, divided by the area of polylinker and fragment of plasmid pCYC117 containing the replicon RA and the gene for resistance to kanamycin, connected together, as shown in figure 3.
2. Recombinant Escherichia coli strain BL21(DE3)/pSVH0106-producer Gl7-acylase.
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, molecular biology, microbiology, genetic engineering.
SUBSTANCE: invention relates to a method for preparing an immunogenic polypeptide inducing immune response that represents the protective response against infection with Bacillus anthracis. Proposed immunogenic polypeptide comprises from one to three domains of the full-scale Protective Antigen (PA) from B. anthracis or their variants and at least one of indicated domains represents domain 1 or domain 4 from PA or its variant. These variants of immunogenic polypeptide and full-scale PA are produced as result of expression in E. coli. Also, invention proposes a vector for expression in bacterial cells that comprises nucleic acid encoding abovementioned immunogenic polypeptide. Also, invention the developed method for prophylaxis of infection caused by B. anthracis based on administration of sufficient amount of immunogenic polypeptide. Also, invention proposes a vaccine for prophylaxis of infection caused by B. anthracis that comprises the effective amount of immunogenic polypeptide and a suitable carrier. Invention provides preparing the effective agent used for prophylaxis of infection caused by B. anthracis.
EFFECT: improved preparing method and valuable properties of polypeptide and vaccine.
22 cl, 5 dwg, 3 tbl, 6 ex
FIELD: biotechnology, in particular gene engineering, pharmaceutical and food processing industry.
SUBSTANCE: DNA sequence (1341 n.p.) encoding fatty acid -desaturase (447 amino acid residue, 57 kD) of nematode Caenorhabditis elegants is isolated and characterized. Obtained DNA-sequence is expressed in bacterium and yeast cells to produce Biologically active enzyme recombinant form. Said recombinant form is capable to catalyze conversion of dihomo-γ-linolenic acid to arachidonic acid and eicosatetraenoate to eicosapentaenoate.
EFFECT: method for large-scale production of polyunsaturated fatty acid.
15 cl, 4 dwg, 1 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention proposes a new recombinant plasmid pR752 (5269 pair bases) comprising genetic construction under control of bacteriophage T5 promoter and encoding a module polypeptide consisting of 6 histidine residues, hemoglobin-like protein of E. coli, modified fragment of large T-antigen SV-40, translocation domain of diphtheria toxin, spacer sequence (Gly-Ser)5 and human epidermal growth factor (6 His-HMP-NLS-Dtox-(Gly-Ser)5-EGF) and designated for the directed transfer of photosensitizers into target-cell nuclei. By transformation of the strain E. coli M15 (rep 4) with plasmid pR752 the recombinant strain E. coli VKPM B-8356 as a producer of new polypeptide vector is prepared. The usage of this new strain is able to enhance the effectiveness of effect of photosensitizers by some orders. Invention can be used in medicinal-biological industry in preparing agents providing the directed transport of photosensitizing agents into tumor cell nuclei.
EFFECT: valuable biological and medicinal properties of polypeptide.
3 dwg, 4 ex
FIELD: biotechnology, genetic and protein engineering.
SUBSTANCE: invention reports construction of plasmid DNA pES6-1 based on plasmid pET22b(+) and DNA fragment comprising a sequence of artificial gene encoding human interferon β-1b providing expression of human recombinant interferon β-1b. Also, the strain Escherichia coli BDEES6 (BL21(DE3)/pES6-1) as producer of human recombinant interferonβ-1b is prepared. Invention provides enhancing yield of human recombinant interferon β-1b. Invention can be used in medicine and pharmaceutical industry.
EFFECT: valuable biological and medicinal properties of strain.
2 cl, 2 dwg, 2 ex
FIELD: biotechnology, biochemistry, enzymes, amino acids, microbiology.
SUBSTANCE: invention relates to a method for producing and preparing L-amino acids, such as L-tryptophan, L-phenylalanine, L-tyrosine and L-histidine that are prepared by culturing the modified microorganism of genus Escherichia with enhanced activity of 6-phosphogluconolactonase. The claimed invention provides preparing indicated amino acids with the high degree of effectiveness.
EFFECT: enhanced yield of amino acids.
15 cl, 9 dwg, 5 tbl, 14 ex
FIELD: biotechnology, biochemistry, enzymes.
SUBSTANCE: invention reports about preparing a polypeptide possessing with the biological activity of enzyme protein-histidine-phosphatase and a polynucleotide encoding this enzyme also. Based on the polypeptide a pharmaceutical preparation is prepared using in pathological states associated with disturbance function of protein-histidine-phosphatase. Also, antibodies showing specificity to sites of indicated polypeptide are prepared. The use of the invention provides study of the N-phosphorylation process, carrying out the diagnosis of pathology of cellular regulation and growth cells and to regulate pathological states associated with disturbance in function of protein-histidine-phosphatase. Invention can be used in medicine and pharmacy.
EFFECT: valuable medicinal properties of enzyme, improved preparing method.
11 cl, 3 tbl, 10 dwg
FIELD: biotechnology, feedstuff production.
SUBSTANCE: method for phytase-containing aqueous liquid includes cultivation of genus Aspergillus microorganism transformed with expression vector, containing phytase gene of said microorganism bond to promoter and/or signal sequence of amyloglucosidase gene. Cultivation is carried out in aqueous medium containing carbon and nitrogen source under conditions, which allow recombinant phytase expression. Cultural liquid is filtered, cation exchange chromatography, anion exchange chromatography, and ultrafiltration are carried out to produce phytase-containing aqueous liquid having activity at least of 14000 U/g. Said aqueous liquid with phytase activity is useful in production of granulated material, animal feed, premix or semi-finished animal feed.
EFFECT: granulated material and animal feed for animal growth stimulation.
24 cl, 2 tbl, 10 ex
FIELD: genetic engineering, biotechnology, biochemistry, medicine.
SUBSTANCE: invention represents a polypeptide of new family of phosphodiesterases and a polynucleotide encoding thereof. Invention relates to the development of methods for detecting partners in specific binding indicated polypeptide and polynucleotide involving stages for their contacting with a compound, detection for binding and detection a compound as a partner for specific binding. Also, invention proposes the constructed expression construction that is used in the method for preparing polypeptide of new family of phosphodiesterases for preparing a cell-producer. Also, monoclonal and polyclonal antibodies raised to this polypeptide have been prepared. Invention describes anti-sense polynucleotide for regulation of expression of polypeptide of new family of phosphodiesterases. Using the invention provides additional pharmacological approaches in treatment of states associated with disturbance of metabolic ways of cyclic nucleotides.
EFFECT: valuable medicinal and biochemical properties of polypeptide.
27 cl, 3 tbl, 11 ex
FIELD: biotechnology, amino acids, biochemistry.
SUBSTANCE: invention relates to a method for preparing proteinogenic or non-proteinogenic L-amino acids, especially L-phosphotricine, from their racemic N-acetyl-D,L-derivatives. Method involves enzymatic cleavage of racemate by using recombinant hyppurate hydrolases that results to selective deacetylation of N-acetyl-L-derivatives of corresponding L-amino acids while N-acetyl-D-derivatives of corresponding D-amino acids are not deacetylated. Prepared deacetylated L-amino acids are separated by preparative method from non-deacetylated N-acetyl-D-derivatives and/or not fully deacetylated N-acetyl-L-derivatives. Invention provides preparing L-amino acids with the high yield and purity of enantiomers.
EFFECT: improved preparing method.
9 cl, 4 tbl, 6 ex
FIELD: biotechnology, in particular production of genetically modified plants with altered level of one metabolite secondary product.
SUBSTANCE: claimed method includes selection of nuclear acid sequence encoding enzyme, which is absent in secondary metabolite process, but is capable to modify utilization of intermediate substrate in secondary metabolite process connected with plant nutrient profile. Then recombinant molecule is constructed, which contains abovementioned sequence, to transform plant cell. From this cell genetically modified plant is regenerated. Obtained plant is useful in animal feed preparation.
EFFECT: plants with high nutrient value and improved quality.
35 cl, 33 dwg, 22 ex
FIELD: biotechnology, biochemistry.
SUBSTANCE: invention represents enzyme that catalyzes reaction for formation of peptide from carboxyl component and amine component. Also, invention relates to strains of microorganism Empedobacter and genus Sphingobacterium that produce this enzyme, and to a method for preparing dipeptides from carboxyl component and amine component using this enzyme. Invention provides synthesis of peptide without carrying out the complex method of synthesis.
EFFECT: improved and easy method of synthesis, reduced cost, high yield of peptide.
11 cl, 18 tbl, 27 ex