Recombinant dna coding fusion protein of human epidermal growth factor fused by glutathione-s-transferase (gst-hegf) sequence and recombinant plasmid pas007 providing gst-hegf synthesis in escherichia coli cells

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to genetically engineered production of human proteins, and may be used for preparing human epidermal growth factor (hEGF) in bacterial cells in the form of glutathione-3-transferase fusion protein. What is constructed is the recombinant DNA coding GST-hEGF fusion protein which consists of an amino acid sequence of glutathione-S-transferase and an amino acid sequence of human epidermal growth factor divided by a cleavage site by enterokinase, and characterised by the nucleotide sequence SEQ ID NO:1. The KpnI/XhoI fragment of the vector pET41 and the above recombinant DNA are used to create the recombinant plasmid pAS007 for expression of GST-hEGF fusion protein in E.coli cells.

EFFECT: invention enables reaching high GST-hEGF expression levels in Ecoli cells.

2 cl, 3 dwg, 1 tbl, 5 ex

 

The invention relates to biotechnology, in particular genetic engineering, and is a recombinant plasmid DNA, causing the synthesis and secretion of epidermal growth factor (human) (hEGF) in the bacterial cells in the form of a hybrid protein with glutathione-S-transferase. The invention can find application in microbiological industry and medicine, in particular, to create new burns and wound healing agents for the diagnosis of malignant diseases associated with the over production of hEGF receptor, as well as in creating new methods of targeted delivery of those or other therapeutic agents into target cells specifically expressing the receptor hEGF.

The level of technology

hEGF - a peptide hormone, produced a variety of tissues and is contained in almost all biological fluids of the human body. hEGF is synthesized as a precursor length 1217 amino acids and undergo further proteolytic processing with the formation of a Mature protein with a characteristic size in 53 and three internal disulfide bonds [1]. hEGF acts on the tissue containing the appropriate receptor on their surface, and stimulates their growth and division. The most potent mitogen in vivo and in vitro hEGF is for epithelial and connective tissue cells. The detail is the first study of the spectrum of effects indicates possible ways for its biotechnological application as wound healing, cytoprotective, anti-ulcer drug, and others [2-7].

To date, known recombinant plasmid DNA, providing the expression of hEGF in secretroom form in the cells of the bacteria Escherichia coli, B.subtilis and yeast S.cerevisiae and Pichiapastoris [8-11]. Despite the known benefits of this approach, namely the correct maturation and folding of recombinant in the process of secretion, protection against intracellular proteases and simplicity and accessibility for further treatment methods, this approach is not without disadvantages, the main factor is the low yield of the target product. In this regard, the improvement of the known methods of production of recombinant hEGF is of great practical interest.

One of the most effective ways to purify the target protein is affinity chromatography [12]. In recent years, widespread methods expression of recombinant proteins in the form of hybrids containing auxiliary polypeptide specifically binding to a compound, which is used as a ligand in the subsequent protein purification by affinity chromatography.

One of the most popular affine domains, widely used for immobilization and purification of the hybrid protein is glutathione-S-transferase (GST). In addition to its high stability and high specificity for attributed the Yu to various sorbents on the basis of glutathione GST is also solubilizers domain. The combination of these characteristics make GST is very attractive for a variety of biotechnological applications.

The prior art known methods for producing biologically active EGF mouse [13] and [14] in the form of their hybrids with GST polypeptide, typically used for the sole purpose of improving the procedure of purification of the obtained product. Both genetic constructs, and recombinant DNA according to the invention is constructed taking into account the possibility of further removal of which is in this case the auxiliary GST polypeptide using post-processing protease, which site is introduced between the two merged sequences (GST and EGF) and in which both the known analogues represented by thrombin. Taking into account the fact that in [14], as in the present invention, a sequence encoding a human EGF (hEGF), it seems preferable to consider as the closest analogue (prototype) of the invention.

Distinctive features of the proposed recombinant DNA from the proposed prototype is the presence in its structure of another site proteasome cleavage (cleavage site by enterokinase instead of the site of cleavage by thrombin), as well as a new nucleotide sequence of SEQ ID N0:1, encoding the hybrid and the resulting RA is the development by the applicant of the specific design, carried out by applying optimal for E. coli codons, structure features combine genes and their transcripts, as well as the necessary adaptation of the resulting sequence to the vector-media. Accordingly, the proposed plasmid pAS007 from plasmid constructions disclosed in the prototype, is distinguished by both its basic elements: another piece related to vector-media and another fragment with the coding sequence (SEQ ID N0:1). The main disadvantage of the proposed prototype of the hybrid protein (and, accordingly, the coding of its sequence) is to use the site of cleavage by thrombin, which is not sufficiently level of specificity, which consequently leads to loss of material (hybrid protein) upon receipt from him hEGF due to the occurrence of side products nonspecific hydrolysis [15]. In contrast, enterokinase for this hybrid sequence was highly specific in relation introduced into the construction site of cleavage.

The disadvantage proposed in the prototype plasmid construction in General is low, the output of the provided expression system of the target product, consisting of not more than 63 mg/l, which with high probability is the result of using weak the th promoter tac, and the lack of adequate optimization and adaptation for embedded system coding sequence.

In the present work, was tasked with securing a new synthetic gene of the hybrid protein GST-hEGF (recombinant DNA) and plasmid DNA with its use, which would not have these disadvantages and provide a higher yield of protein (technical effect), and the possibility of more efficient (no losses associated with non-specific hydrolysis of the use of this protein to produce hEGF (additional results).

Disclosure of inventions

The solution of this problem included the following steps:

a) the design of the structure gene of the hybrid protein, suggesting an optimal combination of its elements from the point of view of expression, purification and subsequent enzymatic cleavage of the hybrid, as well as optimization of the merged sequences; b) receiving and combining the nucleotide sequences encoding the components of the hybrid protein; C) creating a vector constructs for expression of the hybrid protein; d) optimization of conditions for the production and purification of the hybrid protein.

Design, determining the nucleotide sequence of the hybrid gene was designed with preferred .oli codons, features patterns combine the ENES and their transcripts as well as the necessary adaptation of the resulting sequences to their Association and included in plasmid construction.

The nucleotide sequence encoding an optimized version of the gene hEGF with additional "spacer" of 13 amino acid residues, including the site of cleavage by enterokinase, obtained by the method of oligonucleotide synthesis.

To create the expression vector directing in E.coli cells the synthesis of a hybrid protein consisting of GST fused with hEGF was used vector retm-41 [16]. This vector contains the gene for GST under the control of a strong promoter of the gene 10 of phage T7, providing highly efficient expression of foreign genes in E. coli strains expressing the RNA polymerase of phage T7 [16].

Expressing recombinant plasmid pAS007 designed by sublimirovanny obtained previously optimized hEGF gene in the vector retm-41 restriction sites kpni restriction sites/Xho1 (figure 1).

By transforming cells of Escherichia coli strain BL21(DE3) constructed a plasmid pAS007, selection and cultivation of clones transformed with a high level of synthesis of a hybrid protein derived recombinant Escherichia coli strain BL21(DE3)/pAS007 producing a hybrid protein GST-hEGF according to the invention. Synthesis of GST-hEGF in the resulting recombinant strain is under cultivation in conventional selective environments is by adding inducer isopropyl-D-thiogalactoside (IPTG) or lactose and provides an output of a target protein, 200 mg/l of culture.

Thus, the present invention includes 2 object:

The first object is a recombinant DNA that encodes a hybrid protein GST-hEGF, consisting of glutathione-S-transferase and hEGF, separated by a cleavage site by enterokinase, and characterized by nucleotide sequence SEQID No. 1.

A second object of the recombinant plasmid pAS007 for synthesis of a hybrid protein GST-hEGF in Escherichia coli cells and consisting of a DNA fragment with the sequence SEQID No. 1 encoding called hybrid protein, and Cloned/XhoI fragment of plasmid retm-41, connected according to Figure 1.

A brief description of the figures.

Figure 1 - Physical and genetic map of the vector pAS007.

The position of the gene GST-hEGF, site enterokinase EC, other elements of the vectors, unique restriction sites.

Figure 2 - Analysis of gene and specific hydrolysis GST-hEGF using electrophoresis.

Track 1 - GST-hEGF to specific hydrolysis, track 2 - GST and hEGF after hydrolysis specific proteinase-enterokinase, lane 3 - marker molecular mass No. 26610 (ThermoSci., USA) (top-down - 116 kDa, 66.2, 45, 35, 25, 18.4, 14.4), lane 4 - hEGF after the cleanup phase.

Figure 3 Stimulation of proliferation of embryonic fibroblasts mouse line NIH3T3.

Score from increasing the number of cells after 5 days. incubation with EGF in DMEM containing 1% FBS. Initial concentration to etoc 4×103/well. Planting was carried out on 1% FBS for a day before adding EGF (SRB-test). The abscissa shows the concentration of EGF (ng/ml) on the y - axis survival (% of control without added EGF.

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 [17].

Example 1. Design and construction of hEGF gene.

Design options synthetic hEGF gene encoding the Mature polypeptide size 53 ako, merged with 13 amino acid "spacer elements" sequence, which includes the natural site enterokinase, was performed using the program GeneDesigner [18]. This program allows on the basis of amino acid sequences to select the appropriate coding sequence optimized for expression in a selected organism, the host with the characteristic frequency of occurrence of codons and other characteristics that contribute to the efficient heterologous expression of a selected polypeptide. Selected nucleotide sequence of hEGF gene was divided into oligonucleotides with a length of 40 mo using DNAworks [19] for the subsequent Assembly of a gene by the method of recombinant PCR. When designing flanking primers to the x 5 sequences were added to the sequence, contains sites restricts and kpni restriction sites XhoI later used to create expression constructs.

The Assembly of the synthetic gene was carried out in two stages using optimized PCR method for the production of synthetic genes [20] using the oligonucleotides listed in table 1.

Table 1
The oligonucleotides used to assemble the synthetic gene hEGF
No.SequenceP.N.
1CTGGGTACCGGTGGTGG17
2TTTTTGTCGTCATCGTCACCAGAACCACCACCGGTACCCA40
3GGTGACGATGACGACAAAAACTCTGACTCTGAATGCCCGC40
4CAGGCAGTAACCGTCGTGAGACAGCGGGCATTCAGAGTCA40
5ACGACGGTTACTGCCTGCACGATGGTGTTTGCATGTATAT40
6TATTTGTCCAGCGCTTCGATATACATGCAAACACCATCGT40
7GAAGCGCTGGACAAATACGCGTGCAACTGCGTTGTTGGT 40
8TACTGGCAGCGTTCACCGATGTAACCAACAACGCAGTTGC40
9GGTGAACGCTGCCAGTACCGTGACCTGAAATGGTGGGAAC40
10CCCTCGAGGCGCAGTTCCCACCATTTCAGGTC32

In the first stage, 5 µl of each 10-oligonucleotides and conduct stage 1 PCR using Taq polymerase under the following conditions.

Mixture for PCR (100 μl):

10 ál of 10x PCR buffer ("Fermentas");

5 μl of 1 μm of primers No. 1-10

10 μl of 2.0 mm of each dNTP;

30 μl of deionized water;

1 μl of DNA polymerase ("Fermentas").

Conditions for PCR: 94°, 5' (denaturation), 94°, 30"; 50°, 30"; 72°, 10" (amplification).

1 µl of the resulting mixture is used for the second PCR with primers # 1 and # 10 in the same conditions

After re-amplification PCR mixture was analyzed by electrophoresis in 2% agarose gel and identify homogeneous fragment size of about 0.15 TPN. Fragment isolated from the gel using a set of http://WizardPCRPrepsK.it (Promega, USA) according to the manufacturer's instructions.

Example 2. Construction of expression vector pAS007

The obtained PCR fragment with gene hEGF embed in the composition of the vector RET containing the promoter of T7 phage, providing the high expression of heterologous genes in E. coli strains, synthesizing T7 polymerase [21], and the modified gene GST. For this purpose, 100 ng of PCR fragment obtained as described in example 1, hydrolyzing jointly by restrictase Cloned/XhoI and embed using T4 DNA ligase in Cloned/XhoI vector RET.

Received ligase mixture transform competent cells of Escherichia coli strain XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacIqZ□M15 Tn10 (Tetr)] (Stratagene, USA), and the resulting kanamycin-resistant transformants analyzed by PCR screening with primers # 1 and # 10 (table 1) and select "positive" clones forming PCR fragments of 180 BP Several positive clones was checked by sequencing using the same primers T7 and select the clone with the gene GST-hEGF without nonspecific PCR mutation, which is designated as pAS007.

Example 3. The preparation of recombinant E. coli strain - producer GST-hEGF.

The obtained recombinant plasmid pAS007 transform E.coli strain BL21 (DE3) [21] [F-, ompT, hsdSB (rB-, mB-), dcm, gal, Δ (DE3)]. The selection of this strain as recipient for the production GS-hGF due to the fact that it synthesizes the RNA polymerase of phage T7, and also has reduced by activity, thereby increasing the yield of the target protein.

For selection of the most active producer of protein GST-hEGF and selection conditions for the cultivation of individual clones of the transformants grown in 50 the l of LB medium at temperatures from 20°C to 37°C to A600 ~1,0 TH, then make the inductor IPTG to a final concentration of 0.2 mm and continue incubation for 22 hours. Periodically selected aliquots of the cell suspension grown individual transformants and used to determine the growth characteristics of culture and level of production of GST-hEGF bacterial cells. To obtain coarse sediment extract cells destroy ultrasound with periodic cooling in ice (4·30 sec with an interval of 1 min). Cellular debris is removed by centrifugation (13000 rpm, 10 min), received 50 ál of the clarified lysate is used to determine the level of production of GST-hEGF by electrophoresis in LTO-PAG.

A certain level of production of the hybrid protein GST-hEGF in getting the E.coli strain BL21(DE3)/pAS0007 approximately 200 mg of protein per liter of culture producer, which is more than three times the yield of protein according to the prototype [14]. The resulting hybrid protein has an inherent EGF mitogenic activity, although to a significantly lesser extent than EGF.

Example 4. Specific hydrolysis of GST-hEGF

Specific hydrolysis of the hybrid protein GST-hEGF was carried out by making readyready solution special hydrolases - light chain enterokinase [22]. Incubation in the presence of hydrolases continued for 2 hours. In the sequence of amino acid residues hEGF OTS is testout sites nonspecific hydrolysis of enterokinase, therefore, the polypeptide chain of hEGF in the process of hydrolysis is not exposed to enterokinase. Figure 2. it is shown that the hybrid protein hydrolysis is carried out almost completely and results in reaction products - GST and hEGF. When further purified hEGF the product is obtained almost without visible impurities, indicating a high specificity of hydrolysis and is proof that the polypeptide chain of hEGF in the process of hydrolysis is not exposed to enterokinase. Similar high specificity light chain enterokinase person demonstrated previously for specific hydrolysis of the hybrid protein Trx-hEGF [22], where also not detected products nonspecific hydrolysis of hEGF.

Example 5. The biological activity of hEGF

To test the biological activity of hEGF embryonic fibroblasts mouse line NIH3T3 were cultured in plastic culture flasks in DMEM containing 10% FBS and 50 μg/ml gentamycin with CO2-incubator at 37°C in humidified atmosphere containing 5% CO2. Cells subcultured twice a week with a solution of 0.05% trypsin and 0.02% EDTA.

The day before making hEGF fibroblasts were sown in 96-well tablets with a density of 4×103cells/well. The next day cells were washed with DMEM containing no serum was added to fresh DMEM containing 1%FBS, made rEGF and for comparison, a commercially available preparations of mouse EGF (manufactured by Sigma, USA; Serva, Germany) in a concentration range of 0.16-20 ng/ml in increments of 1:2 in three parallel wells for each concentration, and incubated under standard conditions for 5 days. After incubation the number of cells in the wells was determined using the SRB (sul-forhodamine) [23]. The medium from the wells was removed by water-jet pump, the cells were fixed with 10% THU (2 h, +4°C), dried in air, was added to each well 50 μl of 0.4% SRB solution in 1% acetic acid and incubated for 30 minutes at room temperature while mixing on a shaker. Not contacting the protein cells the dye was removed by four washing with 1% acetic acid solution, contacting cells dye after drying was extracted with 100 ál of 10 mM solution nezabvennogo TRIS. The colour intensity was measured by absorption at 540 nm on a plate spectrophotometer. The survival rate of cells for each concentration was evaluated in percentage of untreated control. Based on the data constructed survival curves. Statistical processing of data was performed using the same program. The results are presented in figure 3.

From Figure 2 we can conclude that recombinant EGF has the same biological activity as the commercial counterparts. Moreover, it actively is to be higher than that of EGF production Serva, Germany, and coincides with the activity of EGF production Sigma, USA, in a wide concentration range of 0.16-20 ng/ml.

The list of cited sources

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2. Schultz G, Khaw PT, Oxford K, MaCauley S, Van Setten G, Chegini N Growth factors and ocular wound healing. Eye 1994; 8 (Pt 2): 184-187.

3. Greenhalgh DG. The role of growth factors in wound healing. J Trauma. 1996 Jul 1; 41 (1): 159-167.

4. Schultz G, et al. EGF and TGF-alpha in wound healing and repair. J Cell Biochem. 1991 Apr 1; 45 (4): 346-352.

5. Cristiano RJ, et al. Epidermal growth factor mediated DNA delivery into lung cancer cells via the epidermal growth factor receptor. Cancer Gene Ther. 1996 Jan 1; 3 (1): 4-10.

6. Jinno H, et al. Epidermal growth factor receptor-dependent cytotoxic effect by an EGF-ribonuclease conjugate on human cancer cell lines-a trial for less immunogenic chimeric toxin. Cancer Chemother Pharmacol. 1996 Jan 1; 38 (4): 303-308.

7. Ohno K, et al. Multi-drug delivery system using streptavidin-transforming growth factoralpha chimeric protein. DNA Cell Biol. 1996 May 1; 15 (5): 401-406.

8. Morioka-Fujimoto K, et al. Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli. J Biol Chem. 1991 Jan 25; 266 (3): 1728-1732.

9. Yamagata H, Nakahama K, Suzuki Y, Kakinuma A, Tsukagoshi N, Udaka S Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc Natl Acad Sci USA 1989 May; 86 (10): 3589-3593.

10. Urdea MS, Merryweather JP, Mullenbach GT, Coit D, Heberlein U, Valenzuela P, Barr PJ Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast. Proc Natl Acad Sci USA 1983 Dec; 80 (24): 7461-7465.

11. Clare JJ, Romanes MA, Payment FB, Rowedder JE, Smith MA, Payne MM. Sreekrishna K, Henwood CA Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 1991 Sep 15; 105 (2): 205-212.

12. Sassenfeld HM. (1990) Engineering proteins for purification. Trends Biotechnol., 8 (4): 88-93.

13. Li Wenqing, Xu Bainian, Zhu Jian, Luo Jinxian (1998). Expression of GST-EGF fusion protein in E. coli and its purification. Acta Scientiarum Naturalium Universitatis Sunyatseni, 37, 13-16.

14. ZHI Qing-Wen, LI Qian, WANG Shu-Hao, WANG Yu-Xia, LI Shi-Gui, SUN Man-Ji (2005). Expression of gene of GST-hEGF fusion protein and bioactivity of its product. Chin J Pharmacol Toxicol, 19, 113-117.

15. Davie EW, Kulman JD (2006). An overview of the structure and function of thrombin. Semin Thromb Hemost. 32, : 3-15.

16. Novagen, TB239.

17. Ausubel F.M., Brent R.G., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K.A. Current Protocols in Molecular Biology. Massachusets General Hospital and Harvard Medical School, John Willey & Sons Inc., 1994.

18. Villalobos A, Ness JE, Gustafsson C, Minshull J, Govindarajan S. (2006) Gene Designer: a synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics. 2006 Jun 6; 7: 285.

19. Hoover DM, Lubkowski J (2002). DNA Works: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res. 30(10):43.

20. Dong, Mao R, Li B, Liu Q, Xu P, Li G. (2007) An improved method of gene synthesis based on DNA works software and overlap extension PCR. Mol Biotechnol. 37 (3): 195.

21. Studier, F.W. and Moffatt, B.A. (1986). Use of bacteriophage T7 RNA poymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130.

22. Gasparian M.E., Ostapchenko V.G., Schulga A.A., Dolgikh D.A., Kirpichnikov M.P. Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli. Protein Expression and Purification. Pages pages 133-139. Volume 31, Issue 1, September 2003.

23. Skehan H, Storeng R, Scudiero D. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. N 82 (13) P.1107-1112. 1990.

1. Recombinant DNA encoding a hybrid protein GST-hEGF, which consists of the amino acid sequence of glutathione-S-transferase and amino acid consequently the particular epidermal growth factor (human), separated by a cleavage site by enterokinase, and characterized by nucleotide sequence SEQ ID NO:1.

2. Recombinant plasmid S007 for expression of the hybrid protein GST-hEGF in E.coli cells, consisting of Cloned/XhoI-fragment vector rat and recombinant DNA according to claim 1, connected together as shown in figure 1.



 

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6 cl, 8 dwg, 3 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, specifically to creation of fibroblast growth factor receptor (FGFR) muteins, and can be used in medicine. The polypeptide of the FGFR4 receptor extracellular domain (ECD) acidic region mutein is an FGFR4 ECD chimera or a FGFR4 long acid box version and has more acid residues in the D1-D2 linker region than the wild-type FGFR4 ECD. The muteins may include a point mutation that inhibits glycosylation. The mutein is used to treat a disease associated with one or more FGFR ligands, e.g., proliferative diseases, including various types of cancer, angiogenic disorders and macular degeneration.

EFFECT: invention enables to obtain an FGFR4 ECD acidic region mutein, having low capacity to bind with tissue, by increasing the number of amino acid residues within the D1-D2 linker region.

32 cl, 22 dwg, 11 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically PTH receptor agonists, and can be used in medicine. A polypeptide of formula PTH(1-X)/PTHrP(Y-36) is constructed, where denotes an integer between 11 and 18, and Y=X+1, where PTH(1-X) is an amino acid from 1 to X of the human PTH sequence (SEQ ID NO:5) and PTHrP(Y-36) is an amino acid from Y to 36 of the human PTHrP sequence (SEQ ID NO:6). The polypeptide contains one or more of the following mutations in the PTH(1-X) sequence: Ala in position 1, Ala or Aib in position 3, Gin in position 10, Arg or homoarginine in position 11, Ala in position 12, and Trp in position 14. The obtained polypeptide is used to repair fractures, treat hypoparathyroidism, hyperphosphatemia, tumoral calcinosis, osteoporosis, osteomalacia, arthritis, thrombocytopenia, as well as increase stem cell mobilisation in a subject.

EFFECT: invention enables to obtain a polypeptide having prolonged activity on the PTH receptor.

18 cl, 37 dwg, 8 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: inventions refer to polynucleotide sequence coding the structured pertactin protein (Prn); a vector including such a sequence and compositions containing protein or vector. The characterised polynucleotide sequence codes 300 first amino acids that are the closest to N-end of this type of natural mature Prn (PrnX300), and amino-acid sequence including 620 last amino acids that are the closest to C-end of this type of natural mature Prn (PrnY620) so that structured pertactin PrnX300-PrnY620 of Bordetella class is obtained. Structured molecules Prn include polymorphisms of different B. Pertussis strains and cause immune responses with increased protective ability and opsonophagocytic activity, which exceed the corresponding properties of the preceding vaccines.

EFFECT: protein obtained as per the presented invention can be used in medicine and veterinary as a component of antibacterial vaccines against Bordetella pertusis.

12 cl, 3 dwg, 2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: microorganism-producent is cultivated in a suitable nutrient medium with subsequent extraction and treatment of target protein. At the same time the producent is yeast strain Saccharomyces cerevisiae, transformed with the expression vector, which contains an area of initiation of replication of endogenic 2-mcm plasmid of yeast Saccharomyces cerevisiae, and also a yeast promotor GAL1, which controls gene expression, including a DNA sequence SEQ ID NO: 1, coding a fused protein, the composite parts of which are aminoacid sequences of the protein E7-HSP70 and the protein of ubiquitin of yeast Saccharomyces cerevisiae, occupying within the fused protein an N-end position ending with a processing site, which separates it from the sequence of the protein E7-HSP70 and recognised natural ubiquitin-specific yeast proteinases. The yeast strain Saccharomyces cerevisiae VKPM Y-3853 - producent of the protein E7-HSP70 - is produced by transformation with the expression vector pPDX3U-E7-HSP70 of the yeast strain Saccharomyces cerevisiae D702.

EFFECT: group of inventions provides for high level of biosynthesis and yield of treated protein E7-HSP70, production of a water-soluble correctly processed protein E7-HSP70, principal absence of toxic and pyrogenic bacterial LPS in preparations of the protein E7-HSP70.

2 cl, 4 dwg, 12 ex

FIELD: biotechnologies.

SUBSTANCE: fusion peptide is presented for neutralisation and destruction of organophosphorous compounds, comprising a signal peptide TAT3 with amino acid sequence SEQ ID NO: 5, presented in the description, functionally connected to the sequence of organophosphate hydrolase SEQ ID NO: 18, classified as protein EC 3.1.8. The following components are described: extracted polynucleotide, which codes the specified fusion protein; a vector containing the specified polynucleotide; and a procaryotic host cell containing the specified vector and expressing the specified fusion protein. The method is proposed to produce a ferment, which destroys organophosphorous compounds, including expression of the specified polynucleotide in the procaryotic host cell and production of the specified ferment.

EFFECT: invention makes it possible to increase expression of organophosphate hydrolase in a host cell.

13 cl, 1 tbl, 9 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions refers to biotechnology and concern hypoallergic fused proteins. A presented hypoallergic fused protein consists of one hypoallergic molecule originated from an allergen, fused with a second non-allergic protein originated from a pathogen, or a fragment thereof with the hypoallergic molecule originated from the allergen showing 50% decreased ability to bind IgE, 30% decreased T-cell responsiveness as compared with a wild-type allergen. A nucleic acid molecule coding the fused protein, an expression vector, a host cell expressing the same protein, and a vaccine composition containing the protein are also presented.

EFFECT: presented invention enables preparing therapeutic and prophylactic drugs for an allergy or diseases caused by pathogens with using no toxic adjuvants, showing no side effects.

14 cl, 23 dwg, 17 tbl, 27 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, specifically to creation of fibroblast growth factor receptor (FGFR) muteins, and can be used in medicine. The polypeptide of the FGFR4 receptor extracellular domain (ECD) acidic region mutein is an FGFR4 ECD chimera or a FGFR4 long acid box version and has more acid residues in the D1-D2 linker region than the wild-type FGFR4 ECD. The muteins may include a point mutation that inhibits glycosylation. The mutein is used to treat a disease associated with one or more FGFR ligands, e.g., proliferative diseases, including various types of cancer, angiogenic disorders and macular degeneration.

EFFECT: invention enables to obtain an FGFR4 ECD acidic region mutein, having low capacity to bind with tissue, by increasing the number of amino acid residues within the D1-D2 linker region.

32 cl, 22 dwg, 11 tbl, 18 ex

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