Hybrid protein based on recombinant human erythropoietin, having prolonged action (versions), and method of its production

FIELD: biotechnology.

SUBSTANCE: nucleotide sequences are formed, encoding the hybrid proteins EPO-TR 1.6, EPO-TR 4 and EPO-TR 6. Protein EPO-TR 1.6 is recombinant human erythropoietin fused with a fragment TR 1.6 of the human protein MUC1. Protein EPO-TR 4 is recombinant human erythropoietin fused with a fragment TR 4 of the human protein MUC1. The hybrid protein EPO-TR 6 is recombinant human erythropoietin fused with a fragment TR 6 of the human protein MUC1. Hybrid proteins are produced by the roller cultivation in the suitable conditions the modified mammalian cell line CHO containing a nucleotide sequence encoding the protein with subsequent isolation of the hybrid protein from the culture fluid.

EFFECT: invention enables to produce the hybrid recombinant human erythropoietin having the prolonged action.

4 cl, 4 dwg, 7 tbl, 9 ex

 

The invention relates to the field of biotechnology, in particular to a technology for using genetic engineering methods hybrid proteins of human erythropoietin (EPO), which has high biological activity and prolonged action.

EPO is a factor of growth and terminal differentiation of precursor cells in the bone marrow.

Synthesis of erythropoietin, a hormone that regulates the formation of red blood cells, occurs in the kidney. Circulating in the circulatory system of the EPO binds to a specific receptor precursor cells of erythrocytes, causing their proliferation and differentiation. In a number of pathologies, especially kidney disease, the synthesis of endogenous EPO significantly reduced, which leads to the development of severe anemic conditions. The introduction of recombinant EPO allows you to effectively restore the formation of red blood cells, thereby normalizing the condition of the patients. At the same time, it is known that EPO and other glycoproteins of the plasma, rapidly undergoes enzymatic degradation, deprived of sialic acids, which leads to short circulation time in the body and the need for frequent introduction of protein for maximum clinical effect (J. Pharmacology and Exp. Therapeutics: 327, 2, 308-315; British J. of Cancer: 84, Supp.1, 3-10, 2001). There are several strategies the s stability of EPO in vivo, due to two mechanisms of elimination (excretion) of EPO from the body. It is either increasing the size of the molecule of EPO due to the introduction of additional ligands, resulting in reduced renal clearance (the speed of removing the gun from the body (J.Biol.Chem: 263, 15064-15070, 1988)), or the introduction into the molecule of EPO additional carbohydrate chains, which leads to a greater number of sialic groups and reduces the level of elimination of EPO via expressed in the liver cells (hepatocytes) receptors for Avaliani protein - ASGR (American J. discrimination: 227, 6, 1385-1388).

The EPO molecule has four carbohydrate chains, three of which are associated with a peptide chain through the nitrogen of asparagine (N-glycosylation), and one through the oxygen of serine (O-glycosylation). This glycolysing chains constitute about 40% of the molecular weight molecules (British J. of Cancer: 84, Supp.1, 3-10, 2001). Each of the three N-carbohydrate chains consists of 2-4 branches. For O-carbohydrate chains are characterized by the presence of 1-2 branches. Each of the carbohydrate chains may be terminated by sialic acid residue. The maximum number of sialic acids in the molecule of EPO is 14, which makes the whole molecule of EPO negative charge, deprives it of the opportunity to contact ASGR-receptors of hepatocytes and, accordingly, increases the circulation in the body.

As shown when creating a commercial product based on the EPO Aranesp" to the companies Amgen, introduction into the molecule of EPO two additional sites of N-glycosylation increases the excretion of hormone from the body 3-4 times (Exp. Hematology: 31, 290-299, 2003; US 7,217,689 B1).

To obtain the protein of prolonged action - α-darbepoetin used technology of genetic modification of protein structure EPO. The molecule of the modified EPO (α-darbepoetin) contains 5 substitutions in amino acid sequences, two of which - the replacement of alanine at position 30 and tryptophan at position 88 in the asparagine are additional sites of N-glycosylation. The high degree of glycosylation provides α-darbepoetin prolonged biological activity and clinical effect even when cut in half, compared to EPO, the ability to affine interaction with the specific receptor (US 7,217,689 B1; British J. of Cancer: 84, supp.1, 3-10, 2001).

When creating a hybrid protein of EPO-(P)3, which is the closest analogue of the claimed proteins recombinante EPO with prolonged action, it is shown that the introduction into the molecule of EPO additional sites of O-glycosylation also increases the circulation of the hormone in the body (US 20070190610 A1). In the composition of the hybrid protein of EPO-(P)3 includes three C-terminal peptide of the β-subunit of human chorionic gonadotropin (P), each of which contains four of website O-glycosylamine is (Intern. J. Cell Biology: 2011, ID 275063; US 20070190610 A1). Two P peptide located at the C-end of the molecule of EPO and one on the N-end (US 20070190610 A1). Thus a hybrid protein of EPO-(P)3 is in contrast to EPO 12 additional sites of O-glycosylation, which provides a reduction in clearance and increase in circulation time of EPO-containing protein in the body, compared to EPO. At the same time, the half-life of protein for EPO-(P)3 close to the same parameter α darbepoetin, and the affinity of interaction with the EPO receptor is not reduced.

The task of the claimed group of inventions is to expand the Arsenal of proteins on the basis of recombinant human erythropoietin with prolonged action, and to develop a method of obtaining such proteins.

The problem is solved by obtaining

- hybrid protein EPO-1,6 TR with prolonged action, the corresponding amino acid sequence of SEQ ID NO 4, and represents a recombinant human erythropoietin, fused with a fragment of TR 1,6 protein MUC 1 person;

- hybrid protein EPO-TR 4 with prolonged action, the corresponding amino acid sequence of SEQ ID NO 5, and represents a recombinant human erythropoietin, fused with a fragment of TR 4 protein MUC1 person;

- hybrid protein EPO-TR 6 with prolonged action, suitable what about the amino acid sequence of SEQ ID NO 6, and represents a recombinant human erythropoietin, fused with a fragment of TR 6 protein MUC 1, and

- develop a method of producing a hybrid protein EPO-TR 1.6 or EPO-TR 4 or EPO-TR 6 by roller cultivation in suitable conditions, the modified lines of mammalian cells SNO containing the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 2 or SEQ ID NO 3, followed by separation of the hybrid protein from the culture fluid.

The claimed proteins are characterized by the fact that we have no ligands to the N-terminal site of the EPO molecule, and to create additional sites of O-glycosylation at the claimed proteins as ligand molecules of EPO used a fragment of the human glycopeptide MUC 1. This fragment represents the most glycosylated molecule MUC 1 peptide segment (VNTR), consisting of 20-120 repeats of the peptide TR. Each peptide TR consists of 20 amino acids and contains five potential sites of O-glycosylation (Oncogene: 29, 20, 2893-2904, 2010; Glycobiology: 15 (2), 177-191, 2005). The claimed proteins EPO-1,6 TR, EPO-TR 4 and EPO-TR 6 represent the EPO, merged with the fragments of TR of different length (1,6; 4 and 6, respectively) at the C-terminal site of the EPO molecule.

The construction of strains-producers

Construction of strains-producers consists of the following steps:

1. Obtaining genetic constructs for the expression of the claimed protein EPO-TR 1.6 method of overlapping polymerase goal is Noah reaction (SOE-PCR) using as matrix the structural part of the gene EPO (NP_000790.2) and receipt of genetic constructs for the expression of the claimed protein EPO-TR 4 or EPO-TR 6 by the polymerization of the nucleotide sequence Monomeric peptide TR, containing at the 5' and 3'ends of the restriction sites for endonucleases II-th order BclI and BamHI, which allows to obtain polynucleotide fragments of arbitrary multiplicity.

2. Genetic modification Cho cell line DG44 (Invitrogen, USA), which consists in embedding the DNA site directed integration of the gene of interest (FRT).

3. Construction of expression vectors to obtain the claimed proteins in eukaryotic cells by ligating the genetic structures in the pcDNA5/FRT vector (Invitrogen, USA).

Strains-producers of the claimed proteins Cho DG44 (FRT+/DHFR+)-EPO-1,6 TR No. 1F7, Cho DG44 (FRT+/DHFR+)-EPO-TR 4 No. 1F7 and Cho DG44 (FRT+/DHFR+)-EPO-TR 6 No. 1F7 deposited in Russian national collection of industrial microorganisms (VKPM) and have numbers: PMBC N-136, PMBC H-138 and PMBC H-137, respectively.

The method in General

The strain producing the claimed protein PMBC N-136 or PMBC H-138 or PMBC N-137 grown in adhesion culture in the process roller cultivation. To do this in a roller bottle, 850 square cm2add 250 ml of culture medium, DMEM/F12 (Paneco, Russia)containing 8% fetal serum and 8 mm L-glutamine, and inoculated with cells of the producer strain in the amount of 6.0-8.0×104cells / cm2. The cultivation process should be performed within 14 days in CO2-the incubator with the roller is a stop in the standard for adhesion cell lines conditions: 5% CO 2temperature 37°C, humidity not less than 90% and a speed of 3-4 rpm Replacement of culture medium in a roller bottle on the fresh of the same composition is performed on 3, 5, 7, 9, 11, 14 the day after seeding producer strain. Collected plum culture fluid released from the cells by centrifugation for 20 min at 400 x g and stored at a temperature of - 20°C until use. The yield of the final product - EPO protein-TR 1.6 or EPO-TR 4 or EPO-TR 6 - 20-40 mcg/ml

The selection of the claimed protein from the culture fluid is conducted by the method of affinity chromatography (D. Abercrombie, Allenmark S. and other Affinity chromatography. Methods: ed. World, 1988. - 278 S., Ed. by P. Dean, U. Johnson, F. Milla, Per. s angl. B.A. of Klasickeho). In the first phase collected 1500 ml of the culture fluid is thawed, filtered through a membrane with a nominal cut-off of 0.45 μm (Millipore, USA), add tween-20 to a final concentration of 0.02% by volume and sodium azide to 0.04% by volume. The thus prepared culture liquid slowly put at 4°C in a pre-equilibrated column separate CL - 2B (Sigma, USA), with a volume of 70 ml, immobilized by the method of controllable periodic destruction oxidation monoclonal antibodies EPO-1-3 specific to EPO (RU2451071).

At the end of the sorption and exhaustive washing of the column with 10 mm phosphate buffer with pH 7.4, containing 0.5 M chloride NAT the Oia and 0.02% tween-20, carry out the elution of the protein of 200 mm glycine buffer with a pH of 2.2, containing 1M NaCl and tween-20 at a concentration of 0.02%. The solution lirovannomu protein, volume 70-75 ml, immediately neutralized by adding 700 to 750 ál of 3 M crisologo buffer with a pH of 9.0. The output of the selected protein EPO-TR 1.6 or EPO-TR 4 or EPO-TR 6 is not less than 70%.

Next, perform ion-exchange chromatography selected protein sorbent MonoQ HR5/5 (Pharmacia, Sweden), in accordance with the methods of allocation cialisovernight isoforms of EPO-containing recombinant proteins (US 20090092607; Biotechnology: No. 5, 38-59, 2012). For this purpose, obtained at the first stage of extracting the protein solution concentrated to a volume of 3-5 ml using cell Amicon Ultra-15, when 4000tg for 15 min and 23°C. the resulting concentrate was adjusted to 50 ml of 20 mm Tris-HCl buffer with a pH of 7.3 and again concentrated to a volume of 5 ml in the same way, and then pass the resulting concentrate through a MonoQ HR5/5 at a rate of 0.5 ml/min Then the sorbent was washed with 3 ml of 20 mm sodium acetate buffer with pH 4.5 to remove laboulbene isoforms of the protein EPO-TR 1.6 or EPO-TR 4 or EPO-TR 6 stockli higher than 4.5. Then the sorbent balance 3 ml of 20 mm Tris-HCl buffer with a pH of 7.3 and elute fraction cialisovernight isoforms of the protein EPO-TR 1.6 or EPO-TR 4 or EPO-TR 6 a gradient of ionic strength when 0,22-0,25 M sodium chloride. The yield of the final product at this stage is 80-86% of the applied protein. Received the s thus the protein samples stored at -20°C in 0.05 M Na-phosphate buffer with pH 7.4, containing 0.15 M sodium chloride.

The invention is illustrated by figures 1-4.

Figure 1. Immunochemical characterization of the claimed proteins by the method of Western blot and subsequent manifestation of monoclonal antibodies to EPO (RU2451071) and conjugate goat anti-IgG(mouse) antibodies with peroxidase.

- vertical:

markers of molecular weight from 70 to 10 KD indicated by the arrows to the left of figure 1 (Precision Plus Protein Dual Color Standards, Biorad, USA);

horizontal:

tracks shared proteins, namely:

1 - molecular weight markers;

2 - EPO-TR 6;

3 - EPO-TR 4;

4 - EPO-1,6 TR.

Figure 2-4:

X - days of blood sampling, and measurement:

A) the percentage of reticulocytes among the erythrocytic cells (RET, %),

B) the total hemoglobin in the blood of experimental animals (HGB, g/DL),

C) hematocrit blood of experimental animals (HCT, %).

Y - axis value : RET, % or a NEW g/DL or HCT, % (each point is represented by a mean ± SD with p<0,05)indicated:

- solid line with where there's no shading rectangular marker for α-darbepoetin;

- solid line with circle marker for standard recombinant erythropoietin EPO BRP ("Erythropoietin BRP" (EPO BRP, E1515000));

- broken line to control phosphate buffer containing 0.1% bovine serum albumin.

Figure 2. The measurements of blood experimentality in the period from 2 to 15 days after subcutaneous injection of 30 pmol of the hybrid protein EPO-TR 1.6 in comparison with EPO BRP and α-darbepoetin;

- solid line with shaded rectangular marker for the hybrid protein EPO-TR 1,6;

Figure 3. The measurement results of the blood of the experimental animals during the period from 2 to 15 days after subcutaneous injection of 30 pmol of the hybrid protein EPO-TR 4 in comparison with EPO BRP and α-darbepoetin;

- solid line with shaded rectangular marker for the hybrid protein EPO-TR 4;

Figure 4. The measurement results of the blood of the experimental animals during the period from 2 to 15 days after subcutaneous injection of 30 pmol of the hybrid protein EPO-TR 6 in comparison with recombinant EPO (EPO BRP) and α-darbepoetin;

- solid line with shaded rectangular marker for the hybrid protein EPO-TR 6;

Example 1. Obtaining genetic constructs for the expression of the claimed proteins.

For the expression of EPO protein-TR 1.6 or EPO-TR 4 or EPO-TR 6 in mammalian cells Cho DG44 (FRT+/DHFR+) use the expression vector pcDNA5/FRT (Invitrogen, USA). This vector contains: 1. The CMV promoter, providing a high level of expression; 2. FRT sites for directed integration of the target gene into the genome of cells-producers; 3. The gene of resistance to hygromycin for selection of stable cell strains-producers.

Genetic design for the expression of EPO protein-1,6 TR (SEQ ID NO:1) obtained by the method of overlapping polymerase chain reaction (SOE-PCR) with application as matrix structural part of the gene EPO (NP_000790.2) (Molecular cloning: 3 ed, ext. 13.36, N-Y, 2001), and for protein expression EPO-TR 4 (SEQ ID NO:2) or EPO-TR 6 (SEQ ID NO:3) by the polymerization of Monomeric peptides TR, containing at the 5' and 3'ends of the restriction sites for restriction endonucleases II-th order BclI and BamHI (Bioorganic chemistry, 26, 6, 423-432, 2000) with subsequent legirovaniem in the expression vector pcDNA5/FRT containing the EPO gene.

To confirm the correctness of the structure of the obtained nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 ligated into the cloning vector pUC-57 (Invitrogen, USA)and used to transform cells of the strain E. coli TOP10. The selection of clones of E. coli TOP10 was performed by standard methods on plates with X-gal/IPTG-agar (blue/white test). Next, have used the standard sequencing primers for the vector pUC-57. Then, the sequence encoding the protein is EPO-TR 1.6 or EPO-TR 4 or EPO-TR6, cut by restriction enzymes cut sites, purified in agarose gel and are ligated in the expression vector pcDNA5/FRT.

To obtain genetic structure corresponding to the nucleotide sequence of SEQ ID NO:1, the method of overlapping polymerase chain reaction (SOE-PCR) using primers 1-4 (table 1), (CJSC "Evrogen, Russia).

Table 1
Primers for constructing SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3

The underlined nucleotide sequences 2, 3 and 4 overlap with amplification. Introduced restriction sites are italicized, start codon in bold, the stop codon is marked with an asterisk.
no primerThe nucleotide sequenceRestriction enzymes cut sites
15'-TTT GCT AGC ATG GGG GTG CAC GAA TGT CCT GCC-3'NheI
25'-CAC TGG TCA CGC CAT GTG CTG GCG GTG CGG TTG ATCCGT CCC CTG TCC TGC-3'
35'-TGC GGT TGA TCC CGG TGC CGG ACG GGT ATC TGG AGC ACT GGT CAC GCC AT-3'
4XhoI
55'-TTTT TGA TCA ACC GCA CGC CAG CAC ATG GCG TGA CCA GTG CT-3'BclI
65'-AAA AGG ATC CCG GTG CCG GGG GAC TAT CTG GAG CAC TGG TCA CGC CA-3'BamHI
7 XhoI

To do this, in the first stage, carried out 25 cycles of amplification with the annealing temperature of 47°With the direct primer 1 and reverse primer 2. Obtained and purified in agarose gel product length 622 base pairs (BP) is used for the second stage of amplification with primer 1 and reverse primer 3 (25 cycles with the annealing temperature of 46°C). PCR product 657 BP is used for the third stage PCR with primer 1 and reverse primer 4 (25 cycles with the annealing temperature 65°C). The resulting product SEQ ID NO:1 length of 693 BP phosphorylate using DNA T4 kinase and are ligated according to the blunt ends of the cloning vector pUC-57 restriction site SmaI for later identification. Identified design SEQ ID NO:1 are cut out of the vector pUC-57 sites NheI and XhoI and are ligated in the expression vector pcDNA5/FRT, cut to the same sites.

The polymerization of Monomeric peptide TR for genetic constructs SEQ ID NO:2 or SEQ ID NO:3 is performed with the use of previously obtained vector pUC57-TR1, in which the structure of the peptide is flanked by recognition sites of restricted BclI and BamHI, allowing polymerization of the monomer and to obtain the polynucleotide fragments of arbitrary multiplicity (Bioorgan. chemistry: 26, 6, 423-432, 2000). The resulting restriction of pUC57-TR1Monomeric peptide TR containing the 5' end of the BclI restriction site and 3' end with it enzyme BamHI complete complementary oligonucleotides using primers 5 and 6 or 5 and 7 (Table 1.), receiving, respectively, the nucleotide sequence of TR 1 or TR 2. The sequence TR1 clone in the vector pUC-57, identify, and used for polymerization by successive restrictions - ligating by BclI and BamHI.

Later in this ligase mixture is pre-cut to BclI site and processed alkaline phosphatase plasmid pUC-TR 2 containing the sequence of the Monomeric peptide TR with a stop codon.

Ligase mixture transform cells of E. coli. In subsequent screening select DNA insert containing 4 or 6 repetitions of the peptide TR. Identified DNA containing the sequence of polymers TR 4 and TR 6, cut by restriction enzymes cut sites BclI and XhoI, purified in agarose gel and are ligated in the expression vector pcDNA5/FRT, containing the gene for human erythropoietin, sites BamHI and XhoI.

The result is the nucleotide sequence of SEQ ID NO:1, length of 693 BP or SEQ ID NO:2, length of 828 BP or SEQ ID NO:3, length of 948 BP and the expression vector pcDNA5/FRT, with integrated nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3, designed to receive the strain-producer of the hybrid protein EPO-TR 1.6, the corresponding amino acid sequence of SEQ ID NO:4 or EPO-TR 4 - amino acid sequence of SEQ ID NO:5 or EPO-TR 6, with the amino acid sequence of SEQ ID NO:6, respectively. Estimated molecules of the RNA weight expressed proteins EPO-TR 1,6, EPO-TR 4 and EPO-TR 6, characterized in program Vector NTI.10 (Invitrogen, USA), is 21.2 KD for 25.8 KD and 29.5 KD, respectively.

Example 2. Obtaining genetically modified Cho cell line DG44 (FRT+/HFR+and strains-producers of the claimed proteins.

Genetically modified cell line Cho DG44 (FRT+/DHFR+) is produced by transfection of the cell line Cho DG44 (Invitrogen, USA) vector pFRT/LacZeo/DHFR containing a cassette of genes: gene dehydropeptidase (DHFR), fused to the gene of β-galactosidase and token resistance to zeocin (LacZeo), under the control of the promoter pSV40Δ genes, as well as site-specific homologous recombination for Flp recombinase (FRT).

Vector pFRT/LacZeo/DHFR obtained by ligating the vector pcDNA5(FRT)LacZeo2 bearing FRT sequence for binding of the yeast recombinase and fused gene LacZeo (Invitrogen, USA), the structural part of the gene DHFR mouse, taken from the vector pOptiVEC (Invitrogen, USA), and the promoter region of SV40 vector pCI-neo (Promega).

At the end of step dumartheray selection of the cell line Cho DG44 (FRT+/DHFR+) (resistance to zeocin and restore the phenotype dhfi^) and restore growth characteristics perform the cloning process. To construct strains-producers choose the clone line Cho DG44 (FRT+/DHFR+), with the best results in the transcriptional activity and stability of gene expression lacZeo. Tammy, expressing EPO protein-TR 1.6 or EPO-TR 4 or EPO-TR 6, get in the co-transfection line Cho DG44 (FRT+/DHFR+) expression vector pcDNA5/FRT, with integrated nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 (Example 1), and plasmid pOG44 (Invitrogen, USA)carrying the gene of the yeast recombinase, in the ratio of 1:9. Selection and cloning of the obtained strains was performed at concentrations of hygromycin 600 μg/ml Methotrexate-dependent amplification of the cloned strains is carried out at concentrations of methotrexate 250-1000 μm and a maintenance dose of hygromycin 50 μg/ml of the Content of the claimed proteins in k.zh. evaluate the use of ELISA EPO (Erythropoietin-ELISA-the best, JSC "Vector-best", Russia).

The result is a modified line of mammalian cells Cho DG44 (FRT+/DHFR+and strains of Cho DG44 (FRT+/DHFR+)-EPO-1,6 TR No. 1F7, Cho DG44 (FRT+/DHFR+) - EPO-TR 4 No. 1F7 and Cho DG44 (FRT+/DHFR+) - EPO-1,6 TR NO. 1F7.

Example 3. Expression and purification of the claimed protein EPO-1,6 TR.

To obtain protein EPO-1,6 TR spend roller cultivation of the producer strain Cho DG44 (FRT+/DHFR+) EPO-1,6 TR NO. 1F7. Roller bottles, an area of 850 cm2, seeded with cells of the producer strain in the amount of 4.0 thousand cells / cm2. The cultivation is carried out in the environment DMEM /F12 (Paneco, Russia)containing 8% fetal serum. The volume of the medium in 250 ml. The process roller cultivation is carried out in CO2-incubator with roller installation at 37°C, 5% CO2, humidity >90%) and speed of 3-4 rpm Replacement of culture medium in a roller bottle on the fresh of the same composition is performed on 3, 5, 7, 9, 11, 14 the day after seeding producer strain. Collected plum culture fluid (k.j) released from the cells by centrifugation for 20 min at 400 x g and stored at a temperature of -20°C. Determination of protein content EPO-TR 1.6 in the culture fluid conducting enzyme-linked immunosorbent assay (EPO-ELISA-the best, JSC "Vector-best") (table 2). The protein content of EPO-TR 1.6 in the obtained culture liquid is 29 mg/ml

To highlight the EPO protein-1,6 TR the resulting roller cultivation 1500 ml of the culture fluid is thawed and filtered through a membrane with a nominal cut-off of 0.45 μm (Millipore S.A.). Then add tween-20 to a final concentration of 0.02% by volume and sodium azide to 0.04% by volume.

The selection of protein carry out the method of affinity chromatography. For sorption of protein EPO-TR 1,6 thus prepared culture liquid slowly put at 4°C in a pre-equilibrated column separate CL - 2B (Sigma, USA), with a volume of 70 ml, with immobilized monoclonal antibodies EPO-1-3 specific to EPO (RU251071). At the end of the sorption and exhaustive washing of the column with 10 mm phosphate buffer with pH 7.4, containing 0.5 M sodium chloride and 0.02% tween-20, spend the elution of EPO protein-TR 1,6 200 mm glycine buffer with a pH of 2.2, containing 1M NaCl and tween-20 at a concentration of 0.02%. The solution lirovannomu protein, volume 71 ml, immediately neutralized by adding 710 μl of 3 M crisologo buffer with a pH of 9.0. The yield of protein EPO-1,6 TR after affinity chromatography is 79%.

Next, perform ion-exchange chromatography dedicated EPO-1,6 TR sorbent MonoQ HR5/5 (Pharmacia, Sweden), prepared in accordance with the manufacturer's instructions. For this purpose, obtained in the previous phase protein EPO-1,6 TR concentrated to a volume of 5 ml using cell Amicon Ultra-15, at 4000*g for 15 min and 23°C. the resulting concentrate was adjusted to a volume of 50 ml of 20 mm buffer Tris-HCl with a pH of 7.3 and again concentrated to a volume of 5 ml of the Obtained volume is passed through a MonoQ HR5/5 at a rate of 0.5 ml/min. and Further ion-exchange sorbent was washed with 3 ml of 20 mm sodium acetate buffer with pH 4.5 to remove laboulbene isoforms of the protein EPO-1,6 TR with stockli higher than 4.5.

The content of EPO-TR 1.6 in k.zh., mg/ml
Table 2
The protein content of EPO-TR 1.6 in k.zh. and in the selected medication
Cell line producer strainThe content of EPO-TR 1.6 in the drug after treatment, mg/mlThe molar concentration of EPO-TR 1.6 in a dedicated drug, nm/ml
1234
Cho DG44(FRT+/DHFR+)-EPO-TR 1,629235

Then the sorbent balance 3 ml of 20 mm Tris-LMI buffer with a pH of 7.3 and elute the fraction of acidic isoforms of the protein EPO-1,6 TR at 0.22 M sodium chloride. The volume of the eluate is 4 ml. Concentration of purified protein EPO-TR 1,6 determined by the method of Bradford (BioRad Protein Assay, BioRad, USA). The selected fraction of acidic isoforms of EPO-1,6 TR is 86% of the total amount deposited on MonoQ HR5/5 protein EPO-1,6 TR. The purified protein sample EPO-1,6 TR translate in 0.05 M Na-phosphate buffer solution with pH 7.4, containing 0.15 M NaCl, bringing the concentration of protein in solution up to 2 mg/ml, and stored at -20°C.

Immunochemical properties of protein EPO-1,6 TR characterized by electrophoresis in a 12.5% polyacrylamide gel containing sodium dodecyl sulphate, according to the standard Protocol of laemmli's method with subsequent Western blot. Protein transfer to nitrocellulose membrane with a pore size of 0.45 μm for 30 minutes at 5 mA/cm2 (Biorad, USA). Next, the nitrocellulose with transferred proteins incubated at room temperature for 1 h in a solution containing 3% milk for immunobloting (Biorad, USA), then incubated with monoclonal mouse antibodies to EPO A (RU2451071) for 2 h, with subsequent 3-fold washing phosphate buffer with a pH of 7.2, containing 0.05% tween-20. Immunospecificity the expression of EPO protein-TR 1,6 detected after 2 h of incubation with peroxidase conjugated monoclonal antibodies goat to mouse immunoglobulin (JSC "Sorbent", Russia) in a dilution of 1:3000 using the chromogenic substrate mixture with tetramethylbenzidine (Sigma, USA).

Results identification of protein EPO-TR 1.6 in Western band is presented in figure 1, track 4. The claimed protein is EPO-1,6 TR characterized by a molecular weight of 55 KD, calculated from molecular weight markers (Biorad, USA), exceeding the estimated weight of 21.2 KD (see Example 1), indicating that glycosylamines expressed protein.

Example 4. Expression and purification of the claimed protein EPO-TR 4.

Receiving EPO protein-TR 4 holds as well as protein EPO-1,6 TR (example 3), but using strain Cho DG44 (FRT+/DHFR+) EPO-TR 4 NO. 1F7. The content of protein EPO-TR 4 in the received k.zh. is 20 mg/ml

The selection of protein EPO-TR 4 of k.zh. carried out analogously to the procedure described in example 3, but get 74 ml lirovannomu be the ka, which is neutralized by adding 740 ál of 3 M crisologo buffer with a pH of 9.0. The yield of purified EPO-TR 4 after affinity chromatography is 76%.

The resulting protein solution EPO-TR 4 concentrated to 3 ml using cell Amicon Ultra-15, as in example 3. The concentrate was adjusted to 50 ml of 20 mm buffer Tris-HCl with a pH of 7.3 and again concentrated to a volume of 3 ml of Ion-exchange chromatography dedicated EPO-TR 4 performed similarly to the procedure described in example 3. The selected fraction of acidic isoforms of EPO-TR 4 is 83% of the total amount deposited on a MonoQ HR5/5 protein EPO-TR 4.

Table 3
The protein content of EPO-TR 4 in k.zh. and in the selected medication
Cell line producer strainThe content of EPO-TR 4 in K. J., mg/mlThe content of EPO-TR 4 in the product after treatment, mg/mlThe molar concentration of EPO-TR 4 in the selected drug, nm/ml
1234
Cho DG44 (FRT+/DHFR+)-EPO-TR 420233

Immunochem the ical properties of the protein EPO-TR 4 is determined as described in example 3. Results identification of EPO-TR 4 are presented in figure 1, track 3. The claimed protein is EPO-TR 4 has a molecular mass of 60 KD, calculated from molecular weight markers (Biorad, USA), exceeding the estimated weight of 25.8 KD (see Example 1), indicating that glycosylamines expressed protein.

Example 5. Expression and purification of the claimed protein EPO-TR 6.

Receiving EPO protein-TR 6 spend the same way as in example 3, but using strain-producer Cho DG44 (FRT+/DWR+) EPO-TR 6 NO. 1F7. The content of the hybrid protein EPO-TR 6 in the received k.zh. is 21 ág/ml protein Allotment EPO-TR 6 of k.zh. carried out analogously to example 3, but get 75 ml lirovannomu protein, which is neutralized by adding 750 ál of 3 M crisologo buffer with a pH of 9.0. The output of the EPO-TR 6 after affinity chromatography is 73%.

The resulting protein solution EPO-TR 6 concentrated to 3 ml using cell Amicon Ultra-15, and carry out ion-exchange chromatography dedicated EPO-TR 6, as described in examples 3. The selected fraction of acidic isoforms of EPO-TR 6 is 87% of the total amount deposited on a MonoQ HR5/5 protein EPO-TR 6.

Table 4
The content of EPO-TR 6 in k.zh. and in the selected medication
Cell line strain-p is ducenta The content of EPO-TR 6 in k.zh., mg/mlThe content of EPO-TR 6 in the product after treatment, mg/mlThe molar concentration of EPO-TR 6 in the selected drug, nm/ml
1234
Cho DG44 (FRT+/DHFR+)-EPO-TR 621228

Immunochemical properties of protein EPO-TR 6 characterized as described in example 3.

Results identification of EPO-TR 6 are presented in figure 1, track 2. The claimed protein is EPO-TR 6 is characterized by a molecular weight of 70 KD, calculated from molecular weight markers (Biorad, USA), exceeding the estimated weight of 29.5 KD (see Example 1), indicating that glycosylamines expressed protein.

Example 6. Evaluation of biological activity of the inventive proteins in vitro.

The bioactivity in vitro claimed EPO protein-TR 1.6 or EPO-TR 4 or EPO-TR 6 is determined by their ability to communicate with the EPO receptor and thus to initiate proliferation sensitive to EPO cell line UT-7/EPO. The rate of proliferation of the cell line UT-7/EPO proportional to the amount of the hormone EPO (or its modifications), added to the environment (Blood, 1993, Vol 82, pp 456-464). The speed of prolifer the tion was determined by the colorimetric test. The method is based on the reduction reaction of a colorless tetrazolium salts with the formation of colored compounds - formazan. Recovery of salts tetrazolium occurs only under the influence of the enzymatic activity of living cells - succinate dehydrogenase. The degree of staining is proportional to the number of living cells and allows to measure the degree of cell proliferation (Mosmann T., J.Immunol.Methods, 1983, 65(1-2), P. 55 to 63). As substrate for the enzymatic reaction using the reaction mixture for colorimetric measurement of cell proliferation company Promega (CellTiter 96R AQueous One Solution Cell Proliferation Assay)containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and fenesin methosulfate (PMS) (Promega, Madison, WI, USA).

As standard using recombinant human erythropoietin - EPO BRP.

Cell line UT-7/EPO grown in IMDM medium (Paneco, Russia) supplemented with 2 mm L-glutamine (Paneco, Russia), 10% fetal serum (Invitrogen, USA) and 1 IU/ml of recombinant EPO. Immediately before the test cells replace the culture medium with fresh without adding EPO and incubated for 24 hours for the depletion of the concentration of the hormone EPO in cells. Exhausted thus cells are seated in 96-hole tablet (2*103cells/per well) in 75 μl of IMDM medium with 0.5% fetal serum. Later in t the same wells add 25 ál of the environment, containing from 0 to 500 picomoles (PCM), one of the claimed hybrid proteins or standard recombinant EPO. Tablets with stimulated cells incubated for 72 hours at 37°C in an incubator with 5% CO2. Next to cells add 20 µl of the substrate. Color reaction develops within 2 hours at 37°C, and absorbance (A) was measured at a wavelength of 490 nm.

The calculation of ED50 (table 5), which represents the protein concentration that causes a 50%level of proliferation of sensitive cells, produced in the program GraphPad PRISM.

Table 5
ED50 for standard recombinant erythropoietin EPO BRP and the claimed proteins EPO-1,6 TR, EPO-TR 4 and EPO-TR 6
View EPO-containing proteinED50 (PKM)
EPO BRP18,18±1,09
EPO-TR 1.68,18±1,12
EPO-TR 48,656±1,07
EPO-TR 610,01±1,14

Presented in table 5, the data allow to conclude that all of the claimed proteins capable of interacting with cellular receptors for EPO and call EP is dependent proliferation of sensitive cells UT-7/EPO. The comparison of the ED50 values for standard recombinant EPO and the claimed proteins shows that to achieve the same proliferative effect requires a much larger number of standard recombinant EPO than the claimed proteins.

Thus, the claimed proteins EPO-1,6 TR, EPO-TR 4 and EPO-TR 6 possess functional properties of recombinant EPO man, that is capable of interacting with receptors for EPO and cause cell proliferation in vitro.

Example 7. Evaluation of biological activity of the inventive proteins in vivo.

To assess the biological activity of the hybrid proteins EPO-1,6 TR, EPO-TR 4 and EPO-TR 6 in vivo use as a model strain Balb/c (Brazilian Journal of Medical and Biological Research: 36, 1561-1569, 2003).

Each of the claimed proteins EPO-1,6 TR, EPO-TR 4 or EPO-TR 6 injected subcutaneously group of eight animals. The volume of the introduced protein is 0.5 ml. of the Content of biologically active protein in the preparation is of 0.075 to 0.3 µg in 1 ml of phosphate-saline buffer (pH of 7.2)containing 0.1% bovine serum albumin. This range of concentrations allows to obtain a linear relationship between the number of reticulocytes and a dose of protein. The same buffer solution with albumin injected subcutaneously control animals.

To construct the calibration curve, a separate group of animals injected on 160, 80, 40 what does 20 IU/ml standard recombinant EPO BRP. After 4 days of injection in the blood of experimental animals measure the number of reticulocytes in the blood of animals taken from the orbital sinus into a test tube with ethylendiaminetetraacetic acid (EDTA) (Microvette-200 EDTA, Sarstedt, Germany), and the percentage of the number of Mature erythrocytes (RET %). Quantification of hematopoietic activity of the claimed proteins EPO-1,6 TR, EPO-TR 4 or EPO-TR 6 in comparison with standard recombinant EPO performed using flow hemocytometer analyzer ADVIA 120 (Bayer-Siemens), and program data Multispecies Software 2120.

Table 6
The specific biological activity of the inventive hybrid proteins in comparison with standard recombinant EPO
View EPO-containing proteinThe specific activity of the protein, IU/mg
EPO BRP141120
EPO-TR 1,6648972±37264
EPO-TR 41507278±22722
EPO-TR 62399464±27643

The results presented in table 6, show that biological AK is Yunosti protein EPO-TR 1.6 in four times, protein EPO-TR 4 - ten times, and EPO protein-TR 6 - 15 times higher than the specific biological activity of recombinant EPO. Thus, the claimed hybrid proteins EPO-1,6 TR, EPO-TR 4 or EPO-TR 6 in vivo are characterized by a higher potential stimulation of erythropoiesis than recombinant EPO.

Example 8. Assessment stimulation proesa blood of the claimed proteins in vivo.

To assess the biological properties of the protein E PO-TR 1.6 or EPO-TR 4 or EPO-TR 6 carry out the measurement of key indicators stimulation of hematopoiesis within 15 days after a single subcutaneous injection to a group of 8 experimental animals 0.5 ml of a solution containing 0.05 M Na-phosphate buffer with pH 7.4; 0.1% of bovine serum albumin and 300 RMB protein EPO-TR 1.6 or EPO-TR 4 or EPO-TR 6. The controls are 8 experimental animals after subcutaneous injection of 0.5 ml of a solution containing 300 RMB EPO BRP or 300 RMB α-darbepoetin, and 8 animals after subcutaneous injection of the same buffer, but containing neither one biologically active protein. Daily for 15 days after the start of the experiments in the blood of animal models for measuring the level of reticulocytes (RET), hemoglobin (HGB) and hematocrit (HCT). Measurement of parameters of hemopoiesis is carried out in a flow hemocytometer analyzer ADVIA 120 (Bayer, Siemens, Germany).

The results of the experiment presented in figure 2 and 4 show the presence of eff the KTA stimulate blood proteins EPO-TR 1,6, EPO-TR 4 and EPO-TR 6, which is reflected in the increase in the number of reticulocytes after injection, reaching a maximum on the 4th day (A), the increase of hemoglobin (B) and hematocrit () and lasts for 8-12 days. It should be noted that with the introduction of EPO-1,6 TR, EPO-TR 4 or EPO-TR 6 measured levels of hemoglobin and hematocrit significantly higher than equimolar doses of standard recombinant erythropoietin EPO BRP. In addition, the ability to cause hematopoietic response in proteins EPO-1,6 TR, EPO-TR 4 or EPO-TR 6, in contrast to EPO BRP, persists for at least 8 days, indicating the presence of the claimed proteins prolonged properties. Comparison of experimental results obtained with the introduction of equal doses of EPO-1,6 TR, EPO-TR 4, EPO-TR 6 and α-darbepoetin known high capacity for renewal, shows that during the 7 days of the claimed proteins and α-darbepoetin cause comparable changes in the indices of haematopoiesis, and on the 8th day of α-darbepoetin has a weaker effect on the blood than EPO-1,6 TR, EPO-TR 4, EPO-TR 6.

Thus, the claimed proteins EPO-1,6 TR, EPO-TR 4, EPO-TR 6 have compared with standard recombinant erythropoietin great potential for the correction of disorders, comparable to similar properties of α-darbepoetin.

Example 9. Evaluation of pharmacokinetic properties of the inventive proteins.

Research is the duration of circulation of EPO protein-TR 1.6 or EPO-TR 4 or EPO-TR 6 in vivo spend on line Balb/c mice. Six model animals injected in the tail vein at 0.25 ml of a solution containing 0.05 M Na-phosphate buffer with pH 7.4; 0.1% of bovine serum albumin and 60 RMB/kg of weight of animal protein EPO-TR 1.6 or EPO-TR 4 or EPO-TR 6. The controls are 6 experimental animals after subcutaneous injection of 0.5 ml of a solution containing 60 RMB/kg standard EPO BRP or 60 RMB/kg α-darbepoetin, and 6 animals after subcutaneous injection of the same buffer, but containing neither one biologically active protein. Measurement of levels of all containing EPO protein in the serum of experimental animals to carry through 5, 60, 120, 360, and 480 minutes after injection by ELISA in the system Erythropoietin-ELISA-bis (JSC "Vector-best", Russia), using for each individual protein calibration curves.

To calculate the parameters characterizing the dynamics of circulation and excretion (elimination) of EPO-containing proteins from the organism of experimental animals, using the conventional approach of the kinetic curve of the first order for single chamber models - Ct=C0*exp(-K el*t) (Current Clinical Pharmacology: 1, 5-20, 2006; The Journal of Pharmacology and Experimental Therapeutics: 282, 520-527, 1997),

where Ctthe protein concentration after time t after its introduction experimental animal;

With0the protein concentration at t=0;

To el - constant rate of elimination.

R is the speed of elimination K el, the area under the kinetic curve AUC and half-life t ½ for each of the proteins is carried out by the following formulas:

K el=(In0-In Ct),

AUC=C0/K el,

t½=0,693/K el, where

AUC - value is the area under the pharmacokinetic curve

t½ is the half-life of the protein from the body.

Presents data in table 7 illustrate the differences in the dynamics of circulating EPO protein-TR 1.6 or EPO-TR 4 or EPO-TR 6 compared with standard recombinant erythropoietin EPO BRP and α-darbepoetin in the blood of experimental animals.

As shown in table 7, the half-life of the hybrid proteins of the organism of experimental animals over 2.3 h, corresponding to the time of half-life standard recombinant erythropoietin, and speed off 10 times lower than standard recombinant erythropoietin. EPO hybrid proteins-1,6 TR, EPO-TR 4, EPO-TR 6, in contrast to EPO BRP, have a prolonged properties similar to the properties of α-darbepoetin.

Table 7
Comparative pharmacokinetic parameters of EPO protein-TR 1.6 or EPO-TR 4 or EPO-TR 6 and controls the EPO(BRP) and α-darbepoetin
Dose, nmol/kgKI, nmol/h AUC, nmol*h/mlT½, h
EPO (BRP)600,310,212,3
EPO-TR 1,6600,0911,17,6
EPO-TR 4600,0472,5514,7
EPO-TR 6600,0472,5414.4V
α-darbepoetin600,562,1313

Thus, it is shown that the claimed proteins

- provide stimulation of the formation of precursors of erythrocytes of reticulocytes in experimental animals and possess the ability to effectively adjust the process of blood;

- possess a higher than standard recombinant erythropoietin(EPO BRP), biological activity, calculated by the number of reticulocytes in the blood of experimental animals at 4 days after the introduction of the hybrid protein;

- the level of the HB stimulation of hemopoiesis of the claimed protein is far more efficient than standard recombinant erythropoietin(EPO BRP) and is comparable with the efficiency of α-darbepoetin;

- evaluation of pharmacokinetic parameters shows that the claimed proteins have a prolonged action. At the same time, the circulation in the organism of experimental animals in the hybrid protein EPO-TR 1.6 to 3 times longer than standard recombinant erythropoietin(EPO BRP), and pharmakinetics parameters of the hybrid proteins EPO-TR 4 and EPO-TR 6 is much higher than EPO(BRP) and comparable with parameters α darbepoetin;

- developed a method of obtaining the claimed proteins by culturing engineered strains-producers in suitable conditions, followed by selecting them from the culture fluid by methods of affinity and ion exchange chromatography.

1. The hybrid protein is EPO-1,6 TR on the basis of recombinant human erythropoietin with prolonged action, the corresponding amino acid sequence of SEQ ID NO4, and represents a recombinant human erythropoietin, fused with a fragment of TR 1,6 protein MUC1 person.

2. The hybrid protein is EPO-TR 4 on the basis of recombinant human erythropoietin with prolonged action, the corresponding amino acid sequence of SEQ ID NO5, and represents a recombinant human erythropoietin, fused with a fragment of TR 4 protein MUC1 person.

3. The hybrid protein is EPO-TR 6 on the basis of recombinant erythropoietin che is oweka, with prolonged action, the corresponding amino acid sequence of SEQ ID NO6, and represents a recombinant human erythropoietin, fused with a fragment of TR 6 protein MUC1 person.

4. A method of obtaining a hybrid protein according to items 1 or 2 or 3 by roller cultivation in suitable conditions, the modified lines of mammalian cells SNO containing the coding for this protein the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO2, or SEQ ID NO3, followed by separation of the hybrid protein from the culture fluid.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to muteins of human tear lipocalin, and can be used in medicine. Mutein of human tear lipocalin (hTLc) has identifiable affinity of binding with human receptor Met (c-Met) receptor tyrosine kinase, or its domain, or fragment of human c-Met. Mutein contains from 6 to 18 amino acid substitutions relative to amino acid sequence of mature lipocalin of human tear liquid (SWISSPROT DATABANK ENTRY P31025; SEQ ID NO:36), selected from group, consisting of Arg 26→Thr, Val, Pro, Ser, Gly; Glu 27→Gln, Gly, Val, Ser; Phe 28→Met, Asp; Pro 29→Leu, Ile, Ala, Trp; Glu 30→Leu, Gly, Arg, Phe; Met 31→Ser; Asn 32→Leu, Arg, Val, Gln; Leu 33→Tyr, Val, Ile, Thr, Phe; Glu 34→Val, Arg, Ala; Leu 56→Asn; Ile 57→Gln; Ser 58→Ile, Val; Asp 80→Tyr; Lys 83→Ala; Glu 104→Asp; Leu 105→Thr; His 106→Trp and Lys 108→Gly. Mutein can also additionally contain the following substitutions: Cys 61→Ser; Cys 101→Ser; Cys 153→Ser; Arg 111→Pro; Lys 114→Trp; Thr 37→Ser; Met 39→Ile, Leu; Asn 48→Ser; Lys 52→Thr, Met; Met 55→Leu; Lys 65→Arg, Leu; Ala 79→Leu, Ser; Ala 86→Thr; Ile 89→Ser, Gln, Thr, His; Thr 40→Cys; Glu 73→Cys; Arg 90→Cys; Asp 95→Cys; Lys 121→Cys; Asn 123→Cys and Glu 131→Cys.

EFFECT: invention makes it possible to efficiently treat pathological disorders, which involve pathway HGF/c-Met, as well as to perform identification of human c-Met in sample.

40 cl, 16 dwg, 9 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex.

EFFECT: invention makes it possible to increase output of RTX-toxin ApxI, which can be applied in production of vaccines.

5 cl, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

FIELD: biotechnologies.

SUBSTANCE: invention relates to method for obtaining of RTX-toxins Apxl or ApxIII by culturing of Actinobacillus pleuropneumoniae bacteria in liquid culture media. Characterised method consists in the following: during exponential growth phase of bacteria and production of RTX-toxins air passes through the medium, carbon dioxide content in air is above normal atmospheric level and is up to 10 % vol.

EFFECT: invention allows increasing Apxl or ApxIII toxins output, this may be used during vaccines production.

7 cl, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

7 cl, 13 dwg, 7 ex

FIELD: biotechnologies.

SUBSTANCE: method involves cultivation in the appropriate conditions of yeast Saccharomyces cerevisiae and release of target protein; besides, release is directed with leader polypeptide, which has amino acid sequence SEQ ID NO1 and representing a variant of a pro-area of leader polypeptide of protein PpPIRl Pichia pastoris.

EFFECT: invention enlarges the range of methods for obtaining target protein in yeast Saccharomyces cerevisiae and increases possibilities for effective synthesis of such proteins.

2 dwg, 4 ex

FIELD: biotechnologies.

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4 cl, 5 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

EFFECT: improvement of the method.

5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

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: biotechnology.

SUBSTANCE: hybrid proteins GFN80 and GFN100 are formed based on recombinant human interferon alpha-2 fused on the N-terminus with the amino acid sequence of polypeptide S(G4S)16 or S(G4S)20, respectively. The strains of producer Saccharomyces cerevisiae RNCIM Y-3927 and Saccharomyces cerevisiae RNCIM Y-3928 are produced by recombinant method. The strains are used in the method of production of the hybrid protein GFN80 and GFN100, which comprises culturing under suitable conditions of yeast cells transformed by the expression vector, which contains the region of replication initiation of endogenous 2-micron plasmid of yeast Saccharomyces cerevisiae, and the promoter of yeast GAL1 controlling the expression of the gene comprising the DNA sequence SEQ ID NO:1 or SEQ ID NO:2, respectively, followed by isolation of the hybrid protein from the culture fluid.

EFFECT: invention enables to produce the hybrid recombinant human interferon alpha-2 with the prolonged action in the body of animals.

5 cl, 7 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: there are presented recombinant chimeric polypeptides rmBmpA-frp83, rmOspA-frp83, rmDbpB-rmOspA, rmFlaA-frFlaB and rmOspCBg-rmOspCBa, prepared on the basis of gene expression amplified by PCR on the DNA of Borrelia garinii 20047T Western-Siberian isolate or in case of the protein rmOspCBa, on the DNA of Borrelia afzelii isolate.

EFFECT: invention extends the range of recombinant polypeptides applicable for the serum diagnosis of ixodic tick-borne borreliosis, providing higher specificity and sensitivity of the ITBB, including the differential diagnosis of the early stage and the stage of a disseminated infection in the territories of Borrelia burgdorferi s1 Western-Siberian isolates.

8 cl, 2 dwg, 3 tbl, 5 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

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 biotechnology, in particular to genetic engineering Claimed is artificial gene, which codes chimeric protein of human angiogenin Recombinant plasmid pJZZ-A, representing vector pJexpress414 and containing said gene, embedded on sites NdeI/XhoI, is described Also claimed is recombinant strain of E coli BL21(DE3)/pJZZS-A.

EFFECT: invention makes it possible to increase efficiency of hybrid gene expression and increase production of recombinant chimeric protein of human angiogenin

3 cl, 3 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. What is presented is a vaccine against pneumonia caused by Streptococcus pneumoniae, on the basis of a hybrid protein described by SEQ ID NO:1, containing fragments of the proteins Streptococcus pneumoniae PspA, Sprl 895, PsaA, as well as the ingredients of flagellin as an adjuvant, connected by flexible bridges.

EFFECT: invention provides the effective prevention and therapy of pneumonia ensured by the fact that the vaccine hybrid protein is composed of various immunogenic epitopes eliciting a specific immune response with formed immunological memory.

4 dwg, 8 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: biotechnologies.

SUBSTANCE: there proposed is an antibody specific to TENB2 containing light and heavy chains. Heavy chain contains substitution for free (reaction capable) cysteine A121C that corresponds to A114C (Kabat numbering) or A118C (Eu numbering). Conjugate versions are proposed for prostate cancer treatment containing antibody covalently bound to auristatin, also by means of linker. The following is described: pharmaceutical composition for prostate cancer treatment that uses as active beginning the antibody or its conjugate; product for prostate cancer treatment on the basis of such composition. The invention proposes: method for defining protein TENB2 in the sample - on the basis of antibody as well as analysis for revealing prostate cancer cells at mammal and method for cell proliferation inhibiting on the base of antibody conjugate and auristatin. There described is the method for obtaining antibody conjugate (Ab) and auristatin (D) with expression Ab-(L-D)p, where p is equal from 1 to 4, and L is linker.

EFFECT: invention application provides conjugates with increased stability in serum in comparison with the same conjugates without A121C substitution in antibody that can be used in medicine.

33 cl, 18 dwg, 2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

7 cl, 13 dwg, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to production of peptide derivative mimetic of EPO, with the formula: R1-R2-(CH2)n1-R3-(CH2)n2-R4-R5 (I) and its pharmaceutical salts, where R1, R5 are selected from cyclic peptides with sequences SEQ ID NO:5, 6, 7 and 8; n1, n2 represent integer numbers independently selected from 0-10; R2, R4 are selected from -CO or -CH2; R3 is selected from O, S, CH2, N(CH2)n3NHR6, NCO(CH2)n4NHR6, CHOCONH(CH2)n5NHR6, CHSCON(CH2)n5NHR6 or CHNHCON(CH2)n5NHR6; where n3 represents an integer number selected from 1-10, n4 is an integer number selected from 2-10, n5 represents an integer number selected from 2-10, R6 is selected from H or derivatives of metoxyethylene glycol. The produced peptide or its pharmaceutically acceptable salt is used within a pharmaceutical composition for treatment of disorders characterised with EPO deficit, low or defective population of erythrocytes.

EFFECT: invention makes it possible to produce an agonist of EPO receptor having higher biological activity compared to existing EPO mimetics.

15 cl, 2 dwg, 5 tbl, 19 ex

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