Recombinant plasmid dna pestpc, encoding polypeptide with properties of esterase psychrobacter cryohalolentis k5t, escherichia coli bacteria strain - producer of polypeptide with properties of esterase psychrobacter cryohalolentis k5t
SUBSTANCE: invention relates to biotechnology. Described is a plasmid which codes esterase Psychrobacter cryohalolentis K5T and contains Ndel/Xhol - a DNA fragment of plasmid pET32a with length of 5.366 thousand base pairs, which includes a promoter of bacteriophage T7, a translation enhancer of gene 10 of the bacteriophage T7, a transcription terminator of the bacteriophage T7, a bla β-lactamase gene which determines resistance of cells transformed by plasmid pPC023 to ampicillin, a replication initiation section ori; and Ndel/Sall - a DNA fragment with size of 0.869 thousand base pairs, which contains a gene which codes the mature form of esterase Psychrobacter cryohalolentis K5T with an amino acid sequence SEQ ID NO: 2, given in the description, having an C-end hexahestidine linker. Provided is an Escherichia coli bacteria strain which is modified by said plasmid, a producer of a polypeptide having esterase Psychrobacter cryohalolentis K5T activity.
EFFECT: invention increases the level of biosynthesis of esterase to 20% of total cell protein.
2 cl, 2 dwg, 5 ex
The scope of the invention
The invention relates to biotechnology, particularly genetic engineering. Can be used to obtain the enzyme esterasePsychrobacter cryohalolentisK5Tpossessing high catalytic activity at low temperatures in the reactions of hydrolysis of ester compounds. The esterasePsychrobacter cryohalolentisK5Tcan be used in the food industry for baking industry as additives to the test and in the manufacture of dairy products to accelerate the ripening of cheese; in light industry in the production of household chemicals as components of detergents and spot removers [Hasan, F., A. Shah, et al. Industrial applications of microbial lipases. Enzyme and Microbial Technology. 2006. V. 39(2). P. 235-251].
The level of technology
The main producers of holocausting of esterases are strains ofAspergillus, Fusarium, Mucor, Rhizopussome yeast (Candida) [Coenen, T., P. Aughton, et al. Safety evaluation of lipase derived from Rhizopus oryzae: Summary of toxicological data. Food and chemical toxicology. 1997. V. 35(3-4). P. 315-322; Zhang, N., W. C. Suen, et al. Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. Protein engineering. 2003. V. 16(8). P. 599]. A method of obtaining esterase, based on the cultivation of strains of fungiFusarium oxysporum[P.Rapp. Production, regulation and some properties of lipase activity fromFusarium oxysporumf. sp.vasinfectum. Enzyme and Microbial Technology. 1995. V.17. P. 832-838]. When kultivirovaniya 30 º C for 150 hours maximum accumulation in culture liquid of specific esterase activity 150% act./mg of dry biomass. The disadvantage of this method is the duration of cultivation (6 - 7 days) and the complexity of the procedures for obtaining the culture fluid (exemption from mushroom mycelium).
A method of obtaining holocausting of esterases and lipases by culturing bacteria, such asSerratia marcescens[N. A. Bashkatova, LA Severin. Influence of cultivation conditions on the biosynthesis of lipase fromSerratia marcescens. Microbiology. 1979. So 68, Vol. 5. S. 826],Pseudomonas fluorescens[Dieckelmann, M., L. Johnson, et al. The diversity of lipases from psychrotrophic strains of Pseudomonas: a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens. Journal of applied microbiology. 1998. V. 85(3). P. 527-536],Achromobacter lipolyticum[Khan, I. M., C. Dill, et al. Production and properties of the increasing interest among lipase ofAchromobacter lipolyticum.Biochimica et Biophysica Acta (BBA)-Enzymology. 1967. V. 132(1), p. 68-77]. The use of bacteria has compared with mushrooms advantage in speeding up the process of cultivation. Lack of receipt of esterases by culturing bacteria-producers is a difficult procedure of cleaning and low specific activity of the resulting enzyme.
Another known method of obtaining holocausting lipases and esterases is designing systems expressions of the genes of these enzymes inE. coliusing genetic engineering methods. An example is the system of lipase gene expression Lip1A psychrotrophic Mick is organizma Psychrobacter sp.[Zhang, J., S. Lin, et al. Cloning, expression, and characterization of a cold-adapted lipase gene from an antarctic deep-sea psychrotrophic bacterium,Psychrobacter sp.7195. J Environ Biotechnol. 2007. V. 17(4). P. 604-10] in cellsE. coliBL21(DE3)/ pUC-lipA1, providing access to 0.45 mg of protein from 1 l of culture. The disadvantage of this system is the low yield of the target protein, as well as difficult procedure of cleaning it.
Closest to the claimed method for producing a protein with esterase activity is a method of obtaining protein LipXHiswith lipase activity of psychrophilic bacteriaPsychrobacter sp.C18 [Chen, R., L. Guo, et al. Gene cloning, expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacteriumPsychrobacter sp. C18. World Journal of Microbiology and Biotechnology. 2011. P. 1-11]. The recombinant plasmid pET28a(+)/lipX contains lipase genePsychrobacter sp.C18 under the control of the promoter of bacteriophage T7. Recombinant strain ofE. coliBL21(DE3)/ pET28a(+)/lipX provides protein synthesis LipXHiswith 6 histidine residues at the C-end, which allows its purification using Ni-affinity chromatography. Protein synthesis is carried out with the addition of the inducer isopropyl - β - D - thiogalactopyranoside (IPTG) to a final concentration of 0.2 mm. The result is a protein with lipase activityPsychrobacter sp.C18 with the release of 3.8 mg from 1 l of culture. The disadvantages of the method of obtaining protein LipXHiscan be attributed to the relatively low level of synthesis and not enough high specificactivity.
An object of the invention is to obtain a polypeptide with properties esterasePsychrobacter cryohalolentisK5Tand increase the level of its biosynthesis.
The problem is solved by constructing recombinant plasmid DNA pEstPc that encodes a polypeptide with esterase activity c mol. weight of 4.05 Md (6,235 KBP), consisting of NdeI/XhoI - fragment DNA plasmid pET32a(+) length 5,366 KBP, including the promoter of bacteriophage T7, the transcription terminator of bacteriophage T7 gene bla, β-lactamase, which determines the stability of the transformed plasmid pEstPc cells to ampicillin, plot ori of replication initiation; and NdeI/SalI - fragment DNA size 0,869 KBP containing the gene EstPc encoding the amino acid sequence of the Mature form of esterasePsychrobacter cryohalolentisK5T; containing a unique recognition sites of restriction endonucleases, with the following coordinates: NdeI - 367, XhoI - 840, and also due to the strain of bacteriaEscherichia coliBL21(DE3)/pEstPc producer polypeptide with properties esterasePsychrobacter cryohalolentisK5T.
Recombinant plasmid DNA pEstPc encodes the inducible synthesis of the polypeptide with properties esterasePsychrobacter cryohalolentisK5Tand the strain ofEscherichia coliBL21(DE3)/pEstPc provides a synthesis of this polypeptide with the level of expression of at least 20% of the total cell be is CA. Inducible high-level synthesis of the target polypeptide is provided that plasmid pEstPc contains the promoter of the bacteriophage T7 and the amplifier broadcast of the gene 10 of bacteriophage T7, and a deletion of the 5'end of the gene sequence of the esterasePsychrobacter cryohalolentisK5T,encodes a signal peptide.
A distinctive feature of the proposed plasmid constructions is the presence in its composition of the coding sequence of the Mature form of esterasePsychrobacter cryohalolentisK5T,devoid of the signal peptide (SEQ ID NO 1),under the control of a strong adjustable T7lac promoter, which, combined with the presence of plasmid gene repressor LacI and in the chromosome of the host strain gene RNA polymerase of bacteriophage T7, provides inducible synthesis of the target protein (SEQ ID NO 2) with reliable regulation and high yield, achieved at low concentrations of inducer. Previously when cloning full-esterase genePsychrobacter cryohalolentisK5Twe found that the expression level of this gene in cells ofEscherichia colivery low.The deletion of the fragment of the gene corresponding to the signal peptide, leads to increased levels of synthesis of the recombinant protein in the cytoplasm of bacterial cells. At the 3'end of a gene is a sequence encoding six histidine residues, to facilitate purification of the target white is and using metalroofing chromatography.
To obtain strain-producer of the polypeptide with properties esterasePsychrobacter cryohalolentisK5Tcompetent cellsEscherichia colistrain BL21(DE3) transformed with recombinant plasmid pEstPc.
The technical result of the claimed invention is that the resulting strainE. coliBL21(DE3)/pEstPc allows to obtain a polypeptide with properties esterasePsychrobacter cryohalolentisK5Tnot less than 20% of the total cellular protein at a concentration of inducer 0.1 mm. Unlike the prototype in this strain is achieved in 7.2 times higher yield of the target protein using 2 times less inductor (IPTG). The combination of the above-mentioned properties of the strain ofE. coliBL21(DE3)/pEstPc leads to increased manufacturability of the process of obtaining holocausting esterase.
List of figures
The invention is illustrated graphics.
Fig. 1. Physical map of recombinant plasmid pEstPc. Specified unique sites of restriction endonucleases. T7 promoter - the promoter of bacteriophage T7, T7 terminator - the terminator of transcription of bacteriophage T7, lacI gene lac-repressor, Ap - gene resistance to ampicillin, ori - the site of initiation of replication of plasmids, EstPc - recombinant gene EstPc encoding the amino acid sequence of the Mature form of esterasePsychrobacter cryohalolentisK5T.
Fig. 2. Electrophoregram of listblack producer strain E. coliBL21(DE3)/ pEstPc before (lane 1) and after induction (lane 2) in a 13%polyacrylamide gel (M - protein molecular weight markers); the arrow indicates the polypeptide with esterase activity.
Description. The resulting strainEscherichia coliBL21(DE3)/ pEstPc characterized by the following features.
Morphological signsthe cells are small, rod-shaped, gram-negative, motile, 1 × 3-5 µm, risperadone.
Cultural characteristicsfor the growth on agar LB-medium dominated by small colonies with a diameter of 1-3 mm; round, smooth, translucent, shiny, grey, edge smooth; the pasty consistency. Can meet large colonies with a diameter of 4-5 mm; round, smooth, Matt, edge wavy. Growth in liquid media (LB, minimal medium with glucose) is characterized by the clouding of the environment.
Physiological and biochemical characteristics.The cells of the producer strain can grow in the temperature range 20-42°C, the optimum temperature of growth is 37º. The most favorable for growth pH values are in the range of 6.8 to 7.2. When growing under aerobic conditions a culture can assimilate nitrogen as organic compounds (peptone, tripton, amino acids, yeast extract), and ammonium salts. Carbon is assimilated in the form of carbohydrates, polyhydric alcohols (glycerin), amino acids.
Stable is epostl to antibiotics. Cells are resistant to ampicillin (200 μg/ml), due to the presence of plasmid gene beta-lactamase.
The method, conditions and medium composition for the storage of strain.LB-broth with 15% glycerol at a temperature of - 70aboutIn cryovial.
The invention is illustrated by the following examples.
Example 1. Construction of recombinant plasmid DNA pEstPc.
CulturePsychrobacter cryohalolentisK5Tgrown over night on the Cup with agar medium TSB (Difco, USA)+2% NaCl. 20 μl of the obtained biomass suspended in 50 μl of buffer for Taq polymerase (75 mm Tris-HCl, pH of 8.8, 20 mm (NH4)2SO4, 0,1% Tween 20) and destroy within 15 min by heating in a boiling water bath. After centrifugation 5 min at a speed of 13000 rpm, 5 μl of the obtained supernatant added to the reaction mixture for PCR volume of 50 µl, containing 50 pmol of primer PC231F (SEQ ID NO 3), 50 pmol of primer PC231R (SEQ ID NO 4), 2 mm each of the four dNTP, 5 ál 10x buffer for Pfu DNA polymerase (Fermentas), 2.5 EA. Pfu DNA polymerase and 0.5 EA. Taq DNA polymerase (Fermentas). Polymerase chain reaction (PCR) is conducted as follows: denaturation 1 min, 94°C; annealing - 45 s, 52°C; completion - 45 s, 72°C; the number of cycles - 35. 10 µg of PCR product is treated with restrictase NdeI and SalI (Fermentas) under conditions recommended by the manufacturer of the enzymes, the fragment length 0,869 KBP allocate 1% agarose the first gel. 1 μg of the obtained fragment and 0.2 μg of plasmid vector pET32a(+), linearized by treatment with restrictase NdeI and XhoI, length BP 5366 are ligated for 4 h at 12°C in 10 µl of a solution containing 40 mm Tris-HCl (pH 7.8), 10 mm MgCl2,10 mm dithiothreitol, 0.5 mm ATP and 3 EA. T4 DNA ligase (Fermentas, Lithuania). 5 μl of ligase mixture used to transform competent cellsE. coliXL-1 Blue (Stratagene, USA). Transformants plated on LB-agar containing 100 μg/ml ampicillin.
From grown clones secrete plasmid DNA and treated with restrictase NdeI and XhoI, followed by electrophoretic analysis of the lengths of restriction fragments in a 1% agarose gel. The structure of the cloned gene in the selected clones was confirmed by determining the nucleotide sequence. The result is the target plasmid DNA pEstPc (Fig. 1).
Example 2. Obtaining strain-producer of the polypeptide with esterase activity.
Recombinant plasmid DNA pEstPc transform competent cells ofE. coliBL21(DE3) (Novagen) and after cultivation of recombinant clones on LB-agar with ampicillin (100 μg/ml) at 37aboutTo get producing strains polypeptide with esterase activity.
Example 3. Determination of productivity of the strain-producer of the polypeptide with esterase activity.
To determine the productivity of cellsE. coliBL21(DE3/pEstPc grow when 37º in 5 ml liquid LB medium, containing 100 μg/ml ampicillin, for 16 h on a shaker at 250 rpm/min Obtained nightlife culture in a volume of 100 μl is transferred into 5 ml of liquid LB medium (diluted to an optical density of 0.15-0.20 at a wavelength of 560 nm)containing 100 μg/ml ampicillin and grown to an optical density of 0.8 (wavelength 560 nm) at 37º and stirring of 250 rpm Add IPTG to a final concentration of 0.1 mm, after which the cells are grown for 4 h at 27º and stirring 200 rpm Selected sample culture in the quantity 1 optical unit (wavelength 560 nm) and centrifuged 5 min at a speed of 7000 rpm Precipitated cells suspended in 100 μl of buffer containing 125 mm Tris-HCl, pH of 6.8, 20% glycerol, 3% sodium dodecyl sulphate, 3% mercaptoethanol, of 0.005% bromophenol blue, incubated for 10 min in a boiling water bath, the sample volume of 10 µl analyzed by electrophoresis in a 13% polyacrylamide gel with sodium dodecyl sulfate. After electrophoresis the gel stain using Kumasi R-250. After washing the dye gel scan and carry out mathematical processing of the results using the program Scion Image (Scion Corp., USA). According to scan the contents of esterasePsychrobacter cryohalolentis20% of the total cellular protein. Figure 2 presents electrophoregram lysates of cells of strain-producerE. coliBL21(DE3)/pEsyPc (lanes 1 and 3) in a 13%polyacrylamide is the first gel before (lane 1) and after induction IPTG (track 2) (M - protein molecular weight markers; the arrow indicates the polypeptide with esterase activity).
Example 4. Isolation and purification of the recombinant esterasePsychrobacter cryohalolentis.
Spend the induction of esterase biosynthesisPsychrobacter cryohalolentisin cells of strain-producerE. coliBL21(DE3)/pEstPc IPTG (final concentration 0.1 mm) for 4 h, the Cells centrifuged 15 min at 7000 rpm
1 g of wet biomass induced cells resuspended in 10 ml of buffer (50 mm Tris-HCl pH 8.0; 200 mm NaCl), after which destroy cells by treatment in an ultrasonic disintegrator Branson Sonifier 450, not allowing the temperature more than 10°C. the resulting suspension is centrifuged 15 min at 17000 rpm isolation and purification of the desired polypeptide of the supernatant is performed using Ni-affinity chromatography on a column of Ni-Sepharose FastFlow (GE Healthcare) with a volume of 2 ml in the concentration gradient of imidazole (0.1 - 0.5 M) in buffer (20 mm Tris-HCl pH an 8.0; 200 mm NaCl). Combined fractions containing purified protein and having data on protein electrophoresis purity more than 90%, dialist against buffer (20 mm Tris-HCl pH 8.0; 50 mm NaCl) and sterilized. The average yield of purified so esterase is 28 mg from 1 l of culture, which is 7 times greater than that of the prototype.
Example 5. The determination of the activity of recombinant esterasePsychrobacter cryohalolentisK5T
The esterase activity determined using paranitro the Nile butyrate (Sigma) as substrate [Kulakova, L., A. Galkin, et al. Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability. Biochim Biophys Acta. 2004. V.1696(1). P. 59-65]. To the reaction mixture in a volume of 1 ml containing 50 mm Tris-HCl (pH 8.0), 100 mm NaCl, 0,5% Triton X100, 0,25 mm paranitrophenyl of butyrate (Sigma), add 1,5 µg purified protein, the mixture is incubated for 15 min at 30ºC. 200 μl of the reaction mixture after centrifugation at 13,000 rpm for 5 min digging into the wells of 96-hole tablet (Costar). Measured value of the absorbance at 415 nm on a plate reader Model680 (BioRad). Per unit esterase activity take the amount of enzyme required to release 1 µmol of paranitrophenol for 1 min Specific activity is defined as the number of units esterase activity per 1 mg of protein. General esterna activity of the purified target polypeptide in comparison with the prototype 15 times more, and the specific activity is 1300 edict./mg protein, which is 2 times higher than the specific activity of the prototype.
1. Recombinant plasmid DNA RRs encoding a polypeptide with the activity of esterase Psychrobacter cryohalolentisK5Twith mol. weight of 4.05 Md (6,235 KBP), consisting of NdeI/XhoI - fragment DNA plasmids RETA length 5,366 KBP, including the promoter of bacteriophage T7, power broadcast 10 gene bacterial plankton the Aga T7, the transcription terminator of bacteriophage T7 gene bla, β-lactamase, which determines the stability of the transformed plasmid RRs cells to ampicillin, plot ori of replication initiation; and NdeI/SalI - fragment DNA size 0,869 KBP containing the gene Rsquo encoding the amino acid sequence of SEQ ID NO:2 Mature forms of esterase Psychrobacter cryohalolentis K5Twith C-end exegetically linker.
2. The bacterial strain Escherichia coli BL21(DE3)/pPC023 producing polypeptide with the activity of esterase Psychrobacter cryohalolentis K5T.
SUBSTANCE: SLURP-1 peptide is prepared by transformation of competent cells Escherichia coli BL21 (BE3) by recombinant plasmid DNA pET22b(+)/SLURP-1.
EFFECT: invention provides higher effectiveness of SLURP-1 protein synthesis in cells Escherichia coli.
2 cl, 3 dwg, 1 tbl, 6 ex
SUBSTANCE: disclosed is a plasmid which determines synthesis of alkaline phosphatase CmAP, containing a NcoI/SacI-fragment of plasmid pET-40b(+) (Novagen) and a DNA fragment of 1530 base pairs, which contains a chimeric gene consisting of a CmAP gene structural part which is adapted at the N-end for expression in E.coli cells, and a nucleotide which codes a specific sequence for TEV protease. Described is an E.coli Rosetta(DE3) strain which is transformed by said plasmid, and is a producer of chimeric protein, which contains an amino acid sequence of recombinant alkaline phosphatase CmAP. Disclosed is a method of obtaining recombinant alkaline phosphatase CmAP, comprising steps for: incubating said producer strain in a liquid broth LB for 12 hours at 20°C, depositing bacterial cells by centrifuging, disintegration of the cell suspension in a buffer, centrifuging the extract, chromatography of the supernatant fluid on a column with a metal affinity resin, elution of the protein, concentrating active fractions by ultrafiltration, incubation with protease TEV, concentrating the protein solution and extracting the end product by gel filtration.
EFFECT: improved method.
3 cl, 1 dwg, 3 ex
SUBSTANCE: invention refers to recombinant plasmid DNA pER-Hir coding a hybrid protein capable to autocatalytic breakdown to form [Leul, Thr2]-63-desulphatohirudin, to Escherichia coli to an ER2566/pER-Hir strain - a producer of said protein and a method for producing genetically engineered [Leu 1, Thr2]-63-desulphatohirudin. The presented recombinant plasmid DNA consists of the SapI/BamHI fragment of DNA plasmid pTWIN-1 containing a promoter and a terminator of T7-RNA-polymerase transcription, an amplifier of phages T7 gene 10 translation, β-laktamase (Ap) gene, modified mini-intein Ssp DnaB gene, with an integrated sequence of a chitin-binding domain, and the SapI/BamHI-fragment of DNA containing a sequence of a gene of recombinant [Leul, Thr2]-63-desulphatohirudin-1 containing β-laktamase (Ap) gene as a genetic marker, and unique recognition sites of restriction endonucleases located at the following distance to the left from the site BamHI: Nrul - 186 base pairs, Ndel - 594 base pairs, Xbal - 882 base pairs, EcoRV - 2913 base pairs, Hpal - 2966 base pairs.
EFFECT: inventions allow producing said compound which is used as a drug applied to prevent blood hypercoagulation.
3 cl, 1 dwg, 4 ex
FIELD: biotechnology, molecular biology.
SUBSTANCE: invention proposes a recombinant plasmid DNA providing synthesis of fluorescent mutant protein GFR from Aequorea Victoria in Rec+ strains of E. coli under control of regulatory region of recA gene from Proteus mirabilis. Invention provides the enhanced sensitivity of some factors of chemical nature damaging DNA and damaging factors of physical nature also. Invention can be used food processing, pharmaceutical and chemical industry.
EFFECT: valuable biological properties of plasmid DNA.
2 cl, 12 dwg, 2 ex
FIELD: genetic engineering, biotechnology.
SUBSTANCE: invention proposes a fused protein of beta-galactosidase and cellulose-binding domain of endoglucanase CelD from Anaerocellum thermophilium. The recombinant plasmid DNA pLACspCBD is constructed providing its expression in E. coli cells. The strain E. coli as a producer of indicated fused protein is obtained. A method for preparing this fused protein in immobilized form is developed that involves treatment of cellulose with hydrolyzate of the indicated strain of E. coli. Using the invention provides simplifying processing of dairy foodstuffs. Invention can be used in food processing industry.
EFFECT: valuable properties of strain and protein, improved preparing method.
5 cl, 1 dwg, 5 ex
SUBSTANCE: invention relates to genetic engineering and microbiological industry, as well as a genetic structure for exposing protein on the Yarrowia lipolytica yeast cell wall surface. The disclosed structure has a promoter, a terminator, a signal sequence, a selective marker, a nucleotide sequence which encodes an immobilised protein, and a nucleotide sequence which encodes an amino acid sequence containing an amino acid sequence selected from a group of sequences given in the list of sequences under number SEQ ID NO:1 or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6.
EFFECT: invention simplifies the process of purifying the protein produced by the cell and can be used in microbiological synthesis of proteins.
7 dwg, 11 ex, 1 tbl
SUBSTANCE: agent contains mutant luciferase of glowworms Luciola mingrelica (SEQ ID No:2), recovered from recombinant cells E.coli transformed by plasmid pETL7, luciferin, magnesium sulphate, tris-(oxymethyl)-aminomethane, acetic acid, sodium ethylene aminotetraacetate, dithiotreitol, bovine serum albumin, sucrose and water.
EFFECT: higher sensitivity of ATP test, lower enzyme consumption.
2 cl, 3 dwg, 1 tbl, 4 ex
SUBSTANCE: method involves transformation of a Clostridium acetobutylicum cell by a vector containing: a replicon origin enabling its replication in C. acetobutylicum; a replacement cartridge containing a first marker gene surrounded by two sequences homologous to selected sites around the DNA target sequence, enabling recombination of said cartridge; a second marker gene representing upp counter-selection marker. The cells expressing the first marker gene are selected with the cartridge integrated in their genome. The cells not expressing the second marker gene with the eliminated said vector are selected.
EFFECT: invention enables producing the transformed Clostridium acetobutylicum cell which is genetically stable and marker-free.
31 cl, 6 dwg, 4 ex
SUBSTANCE: there are presented a polynucleotide coding an antibody, an expression vector, a host cell, compositions containing the antibody, and also a method for preparing the antibody and methods for using the antibody.
EFFECT: invention may be used for preparing therapeutic and diagnostic agents to be used for the purpose of detecting the pathological conditions associated with expression or activity of ephrine B2 ligand pathways.
27 cl, 10 dwg, 3 tbl, 6 ex
SUBSTANCE: polypeptide with phytase activity is proposed. The sequences are shown in the description. A polynucleotide coding the said polypeptide is described. The application of the said polypeptide for the use in a feed mix or a feed supplement is described.
EFFECT: allows to increase modified phytase resistance against temperature and humidity.
6 cl, 6 ex, 12 tbl, 10 dwg
SUBSTANCE: disclosed is procariotic recombinant host cell containing heterologous protein of replication activation, which activated condition-dependent replicon origin, and extrachromosomal DNA molecule, containing heterologous therapeutic gene and condition-dependent replicon origin. Also disclosed are method for producing of plasmid containing heterologous therapeutic gene and condition-dependent replicon origin, plasmid representing suicide vector for gene transferring, containing heterologous therapeutic gene and condition-dependent replicon origin.
EFFECT: method for avoiding of non-controlled overexpression of therapeutic gene and resistance genes.
44 cl, 41 dwg, 10 tbl, 14 ex
FIELD: biotechnology, genetic engineering, pharmaceutical industry.
SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.
EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.
2 cl, 3 dwg, 4 ex
SUBSTANCE: invention relates to biotechnology, specifically to obtaining modified IGF-1 proteins and can be used in medicine. Constructed is a polypeptide which contains a human IGF-1 precursor protein, wherein amino acids G1, P2 and E3 are removed as a result of deletion or amino acid E3 is removed as a result of deletion, and wherein amino acid R37 is replaced with alanine and amino acids R71 and R72 are removed as a result of deletion. The cleavage of the E-peptide from IGF-1 by a protease is reduced as a result of said modifications. The obtained polypeptide is used to treat a musculoskeletal disease, diabetes, conditions associated with neuron death, anaemia, chronic obstructive pulmonary disease and burn injury.
EFFECT: invention enables to obtain stabilised polypeptides containing a modified sequence of an IGF-1 precursor, in which the cleavage of the E-peptide from IGF1 which occurs in natural physiological conditions is reduced.
21 cl, 12 dwg, 1 tbl, 82 ex
FIELD: process engineering.
SUBSTANCE: invention relates to biochemistry. Method of producing silver nanoparticles consists in preparing tylosis extract by lavigating tylosis bulk in water with further centrifugation, mixing said extract with silver nitrate, incubating the solution in shaker with further centrifugation, rinsing produced product. Note here that prior to above described process, culture of plant cells is preliminary transformed by agrobacterial vector Agrobacterium tumefaciens GV3101pMP90RK/pPCV002/35S-LoSilAl-nos, containing silicatein gene LoSilAl that ensures biosynthesis of silver nanoparticles sized to 20-80 nm.
EFFECT: efficient process.
1 cl, 6 dwg, 1 tbl, 1 ex
SUBSTANCE: invention discloses a method of identification of the elements which have the ability to terminate the transcripts. To identify the termination sequences a reporter system is used intended for transient transfection in the culture of cells and containing bicistronic matrix. The matrix has the following structure: located under the common promoter one after another open frames of reading the marker proteins of cistron 1 and cistron 2, after the last cistron the known transcription terminator is incorporated; between the cistrons the stop codons are incorporated and a site for internal initiation of translation (IRES); the element tested on the ability to terminate the transcripts is incorporated between the stop codons of the cistron 1 and the site for internal initiation of translation, the gene of resistance to antibiotic, which is located in the body of the plasmid, for selection of cells containing this plasmid.
EFFECT: method can be used for rapid screening of elements for the ability to break off the transcripts and construct the transgenic constructions containing effectively functioning regulatory elements, when creation of the expression vectors in biotechnology, agriculture, medicine.
2 dwg, 3 tbl, 4 ex