Method for large-scale production, separation and purification of recombinant human granulocyte colony-stimulating factor
SUBSTANCE: disclosed is a method of producing, separating and purifying the human granulocyte colony stimulating factor (hG-CSF). Cells of a recombinant producer strain are obtained and the culture fluid is then concentrated. Bacterial cells are broken down while separating inclusion bodies via double treatment with high pressure. Protein impurities are removed. The inclusion bodies are dissolved and protein is recovered. Protein renaturation is carried out for 70-90 hours while diluting the protein solution to 400 ml. The obtained solution is deposited on an anion-exchange chromatographic column with diameter of 300 mm with linear rate of deposition of 600-1000 ml/min. The hG-CSF is then concentrated successively on chromatographic columns with anion-exchange or cation-exchange sorbents. The hG-CSF is then purified on a cation exchanger in fraction recycling conditions and stabilised by dialysis.
EFFECT: method enables to obtain, in one cycle, a stable preparation of hG-CSF in amount of 15-20 g with high homogeneity on HPLC and high hemostimulating activity.
5 cl, 1 dwg, 2 ex
The invention relates to biotechnology, particularly to the production of recombinant granulocyte colony-stimulating factor human (RCG-CSF) on an industrial scale and is a way of getting separation and purification of recombinant human G-CSF as a drug, including medical devices, biomass of bacterial cells containing recombinant plasmid DNA with the gene of G-CSF person.
Granulocyte colony-stimulating factor is the principal regulator of the production of neutrophils. G-CSF is a glycoprotein with a molecular mass of 18-20 kDa, isoelectric point of 5.5, active in Monomeric form (Dr. Ken Alibek 27s lecture (G-CSF). It is produced by many cells: stromal bone marrow cells, endothelial cells, macrophages, granulocytes, fibroblasts, astrocytes (Dr. Ken Alibek).
Functions of G-CSF are: stimulation, differentiation and functional activation of progenitor cells neutrophils; effects on the immune system by regulating the functions of T cells and dendritic cells (Dr. Ken Alibek); effects on the nervous system through the provision of anti-inflammatory activity, inhibition of apoptosis and stimulation of neurogenesis (Solaroglu I, et al, A novel neuroprotectant granulocyte-colony stimulating factor, Stroke 2006).
In medicine is used recombinant G-CSF cagliarini, so non. Its clinical effect is not dependent on the glycosylation of the molecule (Bönig N, Silbermann S, et al. Glycosylated vs non-glycosylated granulocyte colony-stimulating factor (G-CSF) - results of a prospective randomised monocentre study, 2001; Hoglund M. Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) - what is the difference? Med Oncol. 1998 Dec)
Recombinant G-CSF man is widely used in Oncology and Hematology in the treatment of neutropenia different etiology (David C. et al, "A Randomized Controlled Phase 3 Trial of Recombinant Human Granulocyte Colony-Stimulating Factor (Filgrastim) for Treatmentof Severe Chronic NeutropeniaBlood", 1993), autoimmune diseases (Rutella S, et al., Granulocyte colony-stimulating factor: a novel mediator of T-cell tolerance, 2005), myocardial infarction, stroke, HIV infection (Dr. Kuritzkes. Neutropenia, Neutrophil Dysfunction, and Bacterial Infection in Patients with Human Immunodeficiency Virus Disease: The Role of Granulocyte Colony-Stimulating Factor, 2000), neutropenia in the newborn (A Randomized, Placebo-Controlled Trial of Recombinant Human Granulocyte Colony-Stimulating Factor Administration in Newborn Infants With Presumed Sepsis: Significant Induction of Peripheral and Bone Marrow Blood Neutrophilia, 1994).
The growing need for G-CSF, high efficiency in the treatment of a number of diseases of the immune and hematopoietic systems, the high cost of foreign counterparts make the actual task of developing a large-scale method of its production.
The use of microbiological synthesis based on the technology of recombinant DNA molecules provides the ability to scale the process to obtain the target product is a high yield of relatively inexpensive and safe raw materials.
The literature describes several ways to obtain RCG-CSF from cells of E. coli (Welte K. et al. / / Proc. Natl. Acad. Sci. USA, 1985, V.82, No.5, p.1526-1530; H. Nomura et al. // EMBO J., 1985, V.5, No.5, p.891-896; Morioka E. et al. // Res. Exp. Med. (Berl.), 1990, V.190, No.4, p.229-238). However, in some organic solvents, leading to partial denaturation of the protein (Welte K. et al), in other uses expensive reagents, for example, oxidized glutathione (Morioka E et al). A common shortcoming is the low yield of the target protein, and the scaling of the described processes is complicated by the use of chromatography low speeds.
A method of obtaining G-CSF from E. coli cells SG20050 transformed by the plasmid pGGF8 (EN 2201962). Cells were destroyed using ultrasound, the inclusion body was washed and dissolved using 8M urea, renaturation RCG-CSF was performed by diluting a neutral buffer. The chromatographic purification RCG-CSF was performed using one-step ion-exchange chromatography on two serially connected columns with DEAE-cellulose and SP-separate at pH 4.4 with subsequent elution of the target protein from the column with SP-separate in a linear gradient of sodium chloride (0-0 .4 M) in 20 mm sodium acetate buffer at pH 4.4-4.5. The yield of the target product was 24-30 mg from a one liter culture of E. coli cells SG20050/pGGF8. The purified preparation contained 200 ng of bacterial proteins and 80 PCG DNA at 1 mg rhgcsf. The disadvantages of this method include the large loss of the target protein in the preparatory stages at wash NFC, as well as on the main stages due to the low efficiency of the process of renaturation. Get drugs RCG-CSF are characterized by a high content of impurity proteins and DNA producer strain (about 200 ng and 80 PC 1 mg RCG-CSF, respectively).
Closest to the claimed method is a method of obtaining RCG-CSF from E. coli cells SG20050/pGGF8 (EN 2278870), in which the transformed E.coli cells/pGGF8 grown as described previously (EN 2326169), destroy using a French press or ultrasound at a temperature of (6±3)°C. the inclusion Body containing RCG-CSF, separated from the soluble fraction of cell proteins using a laboratory centrifuge. To remove impurity bacterial protein inclusion body washed by a buffer solution with a pH of 7-9, containing detergent and 2 M urea. The precipitate tel enable again separated by centrifugation and dissolved in buffer solution with a pH of 7-9, containing 8 M urea and 10-30 mm 2-mercaptoethanol or 2-5 mm dithiothreitol. Renaturation restored RCG-CSF spend dilution buffer solution with a pH of 7-9, containing detergents, glycerol and EDTA to a concentration of total protein 0.1-0.3 mg/ml at a temperature of (4-20)°C for 20-48 hours or by dialysis in a dialysis tube is ostanovlennogo RCG-CSF against buffer solution with a pH of 7-9, contains detergents, glycerol and EDTA at a temperature of (4-20)°C for 20-48 hours.
After renaturation the resulting solution was oxidized protein is passed through a chromatographic column with a diameter of 50-90 mm anion-exchange sorbent, balanced buffer solution with a pH of 7-9, with a bulk velocity of 5-10 ml/min To the United fractions, not contacting the sorbent, add acetic acid to pH 4-5, and the resulting solution was applied to a chromatographic column with anion exchange and cationogenic sorbents 50 mm in diameter and 25 mm in height with a layer of sorbent 50 and 70 mm, respectively. RCG-CSF elute with cation exchange sorbent in a linear gradient of sodium chloride with a volume rate of 1-2 ml/min Fractions containing the target protein are pooled and cialiswhat in dialysis tubing against a buffer solution with a pH of 4-5. Get drugs RCG-CSF contain 25-33 ng impurity proteins and 10-13 PC DNA producer strain of 1 mg RCG-CSF.
The disadvantages of the prototype method is:
1) the Described method is a laboratory is a laboratory equipment, laboratory centrifuge, chromatography columns small size, FPLC with a maximum flow rate of 10 ml/min etc) with appropriate methods (elution linear gradient, dialyzed in dialysis tubing, etc. and approaches that described makes the process open and discrete. However, direct transfer of the results of retrieval of the target protein from laboratory technologies to the industrial method is not obvious, because the methods of theory of similarity, allowing to obtain criteria zoom in biotechnology unsuitable;
2) Laboratory number of cells involved in the technological process 10 grams, it is not possible to obtain the target protein in a sufficient quantity.
The objective of the invention is the design and search of optimal conditions for large-scale production (15-20 g per cycle), isolation and purification of G-CSF person with a high degree of homogeneity and purity, but also with superior quality, suitable for a medicinal product, including for medical purposes.
The essence of the method consists in the following:
1) the Culture fluid obtained from the stage of cultivation of the producer strain E. coli in the fermenter, a working volume of 250 l, concentrated 10 times, destroy cells using a French press and insoluble fractions of cells (NF) sephirot.
2) To remove impurity bacterial proteins NF washed with buffer solution pH 7-9 containing detergents and urea. NFC again separated by the separation.
3) the Washed precipitate of inclusion bodies dissolved in buffer pH 8.0±0.5, containing Tris HCl, urea and 2-mercaptoethanol in accordance with their 20 hours.
4) Renaturation recovered protein perform 10-fold dilution by adding a solution of denatured protein in chilled buffer. Resaturate ends in 70-90 hours. The percentage of homogeneity denaturirovannogo protein is 60-80%.
5) the Solution denaturirovannogo protein is subjected to additional purification from impurity proteins, passing through a column of anion-exchange sorbent with the speed of 500-700 ml/min, equilibrated buffer solution pH 7-9. In the total volume of not contacting the sorbent fractions lower the pH of the buffer to 4-5. The precipitate formed at a temperature of 10-12°C for 24 hours, separated by membranes.
6) the Concentration of the target protein is performed on serially connected column of anion-exchange and cation-exchange sorbents. The fractions containing RCG-CSF, with homogeneity by HPLC above 80% share. The working volume of the protein solution is reduced to 40-50 times.
7) the Cleaning is performed using cation-exchange chromatography in the mode of recycling fractions to increase the degree of extraction of the target protein and increase its purity and homogeneity by HPLC.
8) joint task faction RCG-CSF diafiltrate on ultrafiltration membranes with a cutoff of 10 kDa.
As detergents are non-ionic detergents Triton X-100, tween-20 or a mixture.
PR is the process of cleaning RCG-CSF is closed. All operations are carried out under sterile conditions using pyrogen-free sorbents and buffer solutions.
As cation exchange sorbent use SP-sepharosa, KM-sepharosa, SO3fractogel.
As the anion-exchange sorbent using DEAE-sephacel, DEAE sepharosa.
Output electrophoretic homogeneous RCG-CSF with a purity of more than 98% 60-80 mg from 1 l of cell culture of the strain E. coli SGK25/ pA3GF (Methods of control medical immunobiologicheskih drugs introduced to the people. HOWTO, MUK 4.1/4.2/588-96, M., 1998). Specific (gemostimuliruyuschee) the activity of the drug received RCG-CSF was determined in vivo in mice, which by the introduction of a cytostatic played hypoplasia blood is 1000-1400%. The specific activity determined in vitro by stimulation of murine cells M-NFS-60 is more than 10+vial size : 8 IU/mg protein. The protein content of the producer strain is 30 ng/mg RCG-CSF. The DNA content is less than 10 PG/mg RCG-CSF (General requirements for medical immunobiological drugs, commissioned by means of genetic engineering. RD 42-68-9-89, M., 1989).
The proposed scheme allows to obtain on an industrial scale stable preparations RCG-CSF with a large quantitative yield, high purity, homogeneity and activity, which exceeds the activity of CSF obtained on a prototype of the u, so you can use them, including as a substance to create a new generation medications.
New compared to the method of the prototype is:
introduction to industrial schemes washing NFC obtaining a generalized pool of washed inclusion bodies; implementation of the laboratory approach of washing of inclusion bodies on an industrial scale possible;
- increasing the number of inclusion bodies involved in biotechnological process to 290-350 g, which corresponds to industrial level production of the protein;
- change the amount and conditions of renaturation in relation to large-scale process:
- increase the volume of the solution of the target protein on stage renaturation of up to 350-450 l
- dilution of the recovered solution tel enable buffer solution pH 7-9 ten times containing EDTA, by making a solution of protein in a total volume of fixed rate with stirring.
- increase time renaturation of 70-90 hours at a temperature of 10-15°C.
- increasing the time of incubation of the protein solution by reducing the pH value of the free fraction after conducting anion-exchange chromatography to 24 hours at 10-15°C.
the concentration of the target protein in 40-50 times with kationoobmennikom in order to reduce the effective volume of the solutions.
- conducting cation-exchange chromatography in riemerella fractions to increase the degree of extraction of the target protein, purity and homogeneity RCG-CSF by HPLC.
- reduce the time of implementation of the process steps by including in the composition of industrial chromatographic systems filters to separate the precipitation in the coating process of protein solutions on the sorbents.
the use of membrane technologies for the stabilization properties of the target protein at the final stage of obtaining the drug substance.
The changes allow you to scale the process of obtaining RG-CSF to the industrial level with preservation of the characteristics yield declared in laboratory method prototype. The total yield of the target protein to homogeneity more than 97% by HPLC is 15-20,
The present invention is illustrated in the figure is a graphic image (see figure 1), which provides:
1. Stage renaturation of 72 hours.
2. The concentration step
3. 1st cycle chromatographic
4. 2nd cycle chromatographic purification
5. 3rd cycle chromatographic purification Examples of specific implementation method below.
(approaching industrial way)
1.1. Receiving and washing of inclusion bodies from 150 grams of cells of the producer strain E. coli SGK25/pA3GF.
Part of the biomass (150 g)obtained in the fermenter with a working volume of 250 liters, suspended in lytic buffer volume of 1000 ml to floor the treatment of homogeneous mass and destroy using a French press at 1100 bar, cooling the cell lysate. The resulting suspension sephirot on the separator. The precipitate is washed with ultrafiltration membranes with a cutoff of 10 kDa in washing buffer containing tween-20, Triton X-100 and urea.
The weight of the resulting inclusion bodies is 20 g, the content of the target protein 72%.
1.2. Solubilization of inclusion bodies. Recovery of protein.
The resulting inclusion body suspended in a buffer solution of pH 7-9 volume of 2500 ml, containing urea, add 2-mercaptoethanol to a final concentration of 20 mm and stirred for 20 hours at room temperature. The formed precipitate was separated by filtration.
1.3. Resaturate protein.
The solution recovered protein was diluted 10-fold cooled to 8°C buffer for renaturation, containing 10 mm sodium phosphate and 10 mm EDTA pH 8.0. Renaturation spend 72 hours at 8°C, formed in the course of renaturation of the precipitate was separated by filtration. The renaturation efficiency assessed by HPLC. Homogeneity denaturirovannogo RCG-CSF by HPLC is 73%.
1.4 Removing impurity bacterial proteins.
The resulting protein solution is applied on a column of HC 45/50 (Pharmacia, Sweden)containing 100 ml DEAE sepharose, balanced buffer Tris HCl, pH is 8.0±0,05, with a speed of 50 ml/min at a temperature of 8°C. Unbound fractions containing RCG-CSF, to collect the target. In a solution of partially purified protein reduce the pH to a value of 4.0±0,05. The solution is incubated for 20 hours at 8°C, the precipitate was separated by filtration.
1.5. Concentration RCG-CSF.
The resulting protein solution is sequentially applied to the column HC 45/50 (Pharmacia, Sweden) anion exchange (DEAE separate (100 ml)and cation-exchange (SO3fractogel (85 ml)) sorbents, balanced acetate buffer with a pH of 4.5±0,05, at a rate of 10 ml/min Target protein elute with SO3fractogel in isocratic mode equilibrating buffer containing 0.9 M sodium chloride. The eluate is collected in a total volume of 1750 ml Homogeneity of the target protein by HPLC is 83%.
1.6. Chromatographic purification on Latinoamerica mode recycle fractions.
A solution of partially purified protein applied to the column with cation-exchanger SO3-fractogel (85 ml) at a rate of 5 ml/min, balanced acetate buffer with a pH of 4.5±0,05. The sorbent was washed with equilibrating buffer containing 0.2 M sodium chloride and protein elute in a gradient of sodium chloride from 0.2 to 1.0 M in 20 column volumes. Fractions with the homogeneity of the target protein by UREH 92% unite.
Combined fractions containing the target protein under the same conditions again put on a column of cation-exchanger SO3fractogel. The sorbent was washed with equilibrating buffer and protein e is irout the same way. Fractions with the homogeneity of the target protein by UREH more than 95% unite as a target product.
1.7. Stabilization of purified RCG-CSF.
Combined fractions cialiswhat against 10 volumes of buffer for dialysis 5 mm sodium acetate pH 4.0. The yield of the target protein 1003 mg of the protein Solution is subjected to sterilizing filtration and stored at 8°C.
2.1. Obtaining cells of the producer strain E. coli SGK25/pA3GF spend on item 1.1., described in Example 1. The culture fluid from the fermenter with a working volume of 300 l was subjected to concentration using membranes to volume of 20-30 liters of cell Suspension twice were processed using a French press at 1200 bar. The content of the target protein in NF is according to gel electrophorese in Page 40-60%.
2.2. The solubilization of inclusion bodies and protein recovery is conducted according to item 1.2. The volume of solution recovered protein is 40 liters
2.3. Renaturation protein is carried out on 1.3. The volume of solution renaturalised protein 400 HP Homogeneity denaturirovannogo RCG-CSF is 70% according to HPLC.
2.4. Removing impurity bacterial proteins is carried out on 1.4. The resulting protein solution is applied on a column of 300 mm in diameter, containing 6000 ml DEAE sepharose with the speed of 600-1000 ml/min at 20°C. Zakislenna the protein solution incubated for 24 hours at 12°C.
2.5. Concentration RCG-CSF is carried out on 15. The resulting protein solution is sequentially applied to the column with a diameter of 300 mm with DEAE separate (6000 ml) and SO3fractogel (6000 ml), with a rate of 200-300 ml/min, the Volume of the eluate 34000 ml. Homogeneity of the target protein by HPLC is 82%.
2.6. Chromatographic purification on Latinoamerica mode recycle fractions spend on 1.6. The protein solution is applied on a column with a diameter of 170 mm with a cation-exchanger Sseparate (5000 ml) at a rate of 200-250 ml/min, balanced acetate buffer. The sorbent was washed with equilibrating buffer. Protein elute in isocratic mode with a solution of 0.13 M sodium chloride. Fractions with the homogeneity of the target protein by UREH 92% unite.
United target protein fractions in the same conditions again put on a column of cation-exchanger. Fractions with the homogeneity of the target protein by UREH more than 95% unite as a target product.
2.7. Stabilization of purified RCG-CSF as the substance conducted according to 1.7. United target fraction is subjected to diafiltration using ultrafiltration membranes with a cutoff of 10 kDa. Protein solution diafiltrate to align its conductivity with the conductivity of the buffer to store protein. The yield of the target protein is 17840 mg of the protein Solution is subjected to sterilizing filtration and stored at 8°C.
Thus, the proposed CSP is about lets get one cycle stable drug RCG-CSF with homogeneity more than 97% by HPLC in the amount of 15-20 grams in 60-100 times more compared with the method of the prototype. The relative protein yield is 60 to 80 mg/ liter of culture liquid of its activity is 1000-1400%, which exceeds the activity values of the prototype, and the duration of the process of obtaining almost match the laboratory technique of G-CSF in the method prototype.
1. Method of production, separation and purification RCG-CSF that includes
a) obtaining cells of the recombinant strain,
b) the destruction of bacterial cells when selecting tel enable,
in preliminary removal of impurity proteins
g) the dissolution of the inclusion bodies,
W) anion-exchange and cation-exchange chromatographic purification
C) and stabilization RCG-CSF
characterized in that exercise concentrating the culture fluid after stage a), two-time processing of high pressure in the destruction of the cells of the producer strain in stage b) with a generalized pool of washed inclusion bodies, solubilization of inclusion bodies, as well as renaturation by dilution of the protein solution to 400 l and duration 70-90 h, anion-exchange chromatographic purification of HC with a diameter of 300 mm with a linear speed of application 600-1000 ml/min, followed by concentration chromatographic columns with anion-exchange and kationoobmena the m adsorbents and chromatographic purification on a cation-exchanger in the recycle mode fractions on stage W) and stabilization RCG-CSF for stage h) dialysis.
2. The method according to claim 1, in which the producer strain is strain of E.coli SGK25/pA3GF deposited in PMBC number In-8685.
3. The method according to claim 1, characterized by obtaining the target protein at the stage of concentrating the solution RCG-CSF by elution in isocratic mode in the presence of 0.9 M To 0.13 M sodium chloride.
4. The method according to claim 1, characterized in that as the cation exchange sorbent during chromatographic purification of the protein in the mode of recycling fractions use Fractogel SO3.
5. The method according to claim 1, characterized in that as the cation exchange sorbent during chromatographic purification of the protein in the mode of recycling fractions using CM sepharose.
SUBSTANCE: imiquimod or resiquimod is injected locally to a mammal over 12-26 hours after injection of nucleotide sequences encoding granulocyte-macrophage colony-stimulating factor and antigen peptide or protein.
EFFECT: significant amplification of immune response of mammal to antigen.
18 cl, 23 dwg, 1 tbl, 1 ex
SUBSTANCE: invention relates to field of biotechnology, namely, to obtaining genetically modified cell lines and can be applied in medicine for immunotherapy and immuno-prophylaxis in patients with malignant neoplasms. By means of recombinant method line of cells of human melanoma KG is obtained, which secretes recombinant granulocytic-macrofagal colony-stimulating human factor. Obtained line is deposited with Specialised cell culture collection of vertebrates of Russian cell culture collection under number RCCC ("П") 699"Д".
EFFECT: line of human melanoma cells KG possesses stable cultural, morphological and immunological characteristics and possesses ability to secrete recombinant human GM-CSF, remaining after cell inactivation with ionising irradiation.
FIELD: gene engineering.
SUBSTANCE: invention can be used for production of recombinant polypeptide of human granulocyte colony-stimulating factor. Recombinant plasmid DNA is constructed in vitro. It includes synthetic gene of human granulocyte colony-stimulating factor, strong constitutive promoter A3 from the early stage of bacteriophage T7 and synthetic section - translation enhancer (TREN) of gene 10 in bacteriophage T7. This DNA in combination with high copy number of plasmid and optimisation of cultivation conditions ensures constitutive biosynthesis of target protein in transformed by this DNA race of Escherichia coli SGK25/pA3GF with high yield.
EFFECT: constitutive biosynthesis of target protein in cells; high yield.
2 cl, 4 dwg, 7 ex
FIELD: medicine, endocrinology, pharmacy.
SUBSTANCE: invention proposes an agent eliciting antidiabetic activity. Agent represents nonglycosylated recombinant human granulocyte colony-stimulating factor. It differs from native preparation by absence of glycosyl group and the presence of additional methionine residue by its N-end. Effect of agent is associated with mobilization and migration of bone marrow mesenchymal stem cells, homing into pancreas. This results to reparation of insulin-producing activity of organ and normalization of the peripheral blood glucose level after monotherapy with recombinant human granulocyte colony-stimulating factor.
EFFECT: valuable medicinal property of agent.
3 tbl, 2 dwg, 1 ex
FIELD: molecular biology.
SUBSTANCE: invention relates to isolated DNA fragment encoding horse GM-CSF, and isolated horse GM-CSF protein. Also disclosed are vectors and various compositions containing thereof. GM-CSF is useful as adjuvant for horse vaccination as well as non-specific immunity stimulator in veterinary.
EFFECT: new compositions for gorse vaccination.
18 cl, 2 dwg, 1 tbl, 7 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention describes construction of recombinant plasmid DNA pFGM17 encoding constitutive synthesis of polypeptide of human granulocytic-macrophagal colony-stimulating factor (GM-CSF) and consisting of Kpn I/Eco RI-fragment of plasmid pSPF1 DNA and artificial DNA sequence encoding signal peptide of the protein Caf1 from Yersinia pestis, and also Kpn I/Eco RI-fragment of intermediate plasmid pSK-GM comprising the synthetic human gene GM-CSF. Escherichia coli cells are transformed with plasmid DNA pFGM17 and strain E. coli BL21(DE3)/pFGM17 is prepared that is a producer of human polypeptide GM-CSF. Invention provides enhancing technological effectiveness and economy of process for preparing recombinant FM-CSF due to excluding the induction stage in biosynthesis process and in simultaneous increasing the yield of the end product by 2 times. Invention can be used for preparing human granulocytic-macrophagal colony-stimulating factor.
EFFECT: valuable properties of plasmid DNA and microorganism strain.
2 cl, 4 dwg, 4 ex
FIELD: genetic engineering, in particular production of human granulocyte colony-stimulating factor.
SUBSTANCE: Recombinant plasmid DNA pES3-7 with molecular weight of 3.63 MDa (5907 b.p.) is constructed. Said DNA consists DNA Ndel/Notl-fragment containing sequence of recombinant G-CSF artificial gene, β-lactamase gene; and plasmid pET22b(+) DNA Ndel/Notl-fragment containing promoter and terminator of T-RNA-polymerase transcription, amplifier of 17 phage 10 gene translation. Plasmid pES3-7 contains as genetic marker β-lactamase gene which determines resistance of E.coli cells transformed with plasmid pES3-7 to ampicillin, and unique restriction endonuclease recognition sites existing on the next distance to the right from Ndel-site: Xbal - 38 b.p.; Hpal - 1332 b.p.; Pstl - 4065 b.p.; Pvul - 4190 b.p.; Xhol - 5363 b.p. Obtained plasmid is used in transformation of Escherichia coli cells to produce strain E.coli BL21(DE3)/pES3-7 as subproducer of recombinant G-CSF. Method of present invention makes in possible to produce recombinant G-CSF with high yield (20-30 % based on total cell protein content).
EFFECT: simplified method for production of recombinant G-CSF with high yield.
2 cl, 2 dwg, 2 ex
SUBSTANCE: method involves conjugation of r-metHuG-CSF with PEG-aldehyde at the free amino group at the N-end of r-metHuG-CSF in the presence of a reducing agent in a buffer solution for pegylation, which contains a polyol of formula CnH2n+2On, where n equals 3-6, or carbohydrate or derivative thereof, where concentration of said polyol or carbohydrate or derivative thereof is in the range of 0.1%-10%, wt/wt.
EFFECT: high output of the r-metHuG-CSF pegylation reaction.
15 cl, 12 dwg, 1 tbl, 10 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, particularly to producing PEGylated granulocyte colony-stimulating factor (G-CSF), and is applicable in medicine. What is produced is a conjugate of G-CSF and monomethoxypolyethylene glycol attached to N-terminal methionine of the G-CSF of general formula: wherein n is integers within 681 to 1 000; m represents an integer ≥4; NαH-G-CSF represents the granulocyte colony-stimulating factor.
EFFECT: produced conjugate exhibits human G-CSF activity and prolonged biological action that makes it possible to use it in pharmaceutical compositions, and as a drug preparation for preventing and treating neutropenias.
17 cl, 10 dwg, 5 tbl, 21 ex
SUBSTANCE: invention refers to biotechnology and genetic engineering, namely to a genetically engineered construct pGoatcasGCSF for human granulocyte colony-stimulating factor (GCSF) expression . The offered invention can be used for producing transgenic animals that are human granulocyte colony-stimulating factor producers. It involves creating the genetically engineered construct pGoatcasGCSF of the size 6386 bps, and a specified nucleotide sequence shown in SEQ ID 1. The genetically engineered construct pGoatcasGCSF includes a 5'-regulatory sequence of the goat CSN1S1 asi-casein gene of the size 3387 bps connected with the full-size human GCSF gene of the size 1485 bps, and a 3'-flanking region of the cow gene CSN1S1 of the size 1514 bps.
EFFECT: stable effective level of human GCSF expression in milk of the transgenic animals, eliminated possibility of ectopic transgenic expression.
9 dwg, 2 tbl, 6 ex
SUBSTANCE: invention relates to granulocyte colony-stimulating factor peptide conjugates formed by linkage through an intact glycosyl linker group of (polyethylene glycol) PEG fragments. Said conjugates are obtained from glycosylated and non-glycosylated peptides under the effect of glycosyl transferase. Glycosyl transferase links a fragment of modified sugar to an amino acid or glycosyl peptide residue.
EFFECT: improved method.
35 cl, 24 dwg, 2 tbl, 6 ex
SUBSTANCE: extraction is done using affinity chromatography with immobilised metal. The method can be realised in native conditions. Biologically active G-CSF is obtained with purity over 95%. Two more chromatography stages are done, cation-exchange and gel filtration, to remove trace amounts of impurities. The method allows for obtaining G-CSF with high output and over 99% purity.
EFFECT: described method is especially suitable for industrial production of G-CSF.
16 cl, 5 dwg, 5 ex
SUBSTANCE: the invention relates to producing new peptides and may be used for treatment and prophylaxis of cytokine-sensitive disorders. Peptides, having a size of 5 to 40 amino acids and arising from cytokines, are used in a vaccine for treatment and prophylaxis of autoimmune diseases, disseminated sclerosis, rheumatoid polyarthritis, psoriasis, autoimmune diabeteses, lupus, allergy, asthma, cancer and AIDS.
EFFECT: allows effective immunization of patients against said diseases while minimizing side effects.
11 cl, 2 dwg, 17 tbl
FIELD: biotechnology, transplantation, in particular, isolation of hematopoietic stem cells.
SUBSTANCE: Lin-negative or weak positive cells derived from bone marrow are cultivated in presence of macrophage colony-stimulating factor (M-CSF) at concentration of approximately 5-20 ng/ml together with cytokine composition including stem cell factor (SCF), interleukin-11 (IL-11) ligand such as fms-like tyrosine kinase (FLT) or thrombocytopoietin (TPO), wherein cytokines are used in the next weight ratio: 1-4 M-CSF:20 SCF:20 IL-11:20 (FLT or TPO).
EFFECT: effective method for targeted propagation of hematopoietic stem cells.
4 cl, 3 dwg, 1 ex
SUBSTANCE: polypeptides for the prevention or treatment of myocardial hypertrophy correspond to sequences of amino acids SEQ ID NO:2-3, 5-8. The composition for prevention or treatment of myocardial hypertrophy comprises a polypeptide corresponding to sequences of amino acids SEQ ID NO:2-3, 5-8, and pharmaceutically acceptable additives. The method of production of the said polypeptides comprises a step of synthesis of the polypeptide with the amino acid sequence SEQ ID NO:2-3, 5-8 in a synthesizer of polypeptides or ligation of the corresponding nucleotide sequence with the vector to form a recombinant vector, transformation of the said recombinant vector into a host cell, induction of expression of the said polypeptide in the said host cell, and isolating of the said polypeptide.
EFFECT: invention enables to produce new polypeptides for prevention or treatment of myocardial hypertrophy.
12 cl, 6 dwg, 9 tbl, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions refers to biotechnology. What is described is a protein-polypeptide complex (PPC) possessing tissue specific regenerative-reparative and rejuvenating action on skin tissue, produced of extracted homogenate of nerve and skin tissues of farm hoofed embryos, a method for preparing it and a pharmaceutical composition thereof. The PPC contains tissue-specific negative faintly acid neutral proteins and peptides with molecular weight 0.5 to 200 kDa with min. 70% of total protein weight having molecular weight falling within the range of 20 to 180 kDa. The BPC is used as an active ingredient in pharmaceutical compositions applicable for preparing therapeutic and cosmetic products.
EFFECT: invention enables producing the preparations having high biological activity and applicable for treating autoimmune, cardiovascular, traumatic, toxic skin diseases, as well as used in aesthetic medicine and cosmetology.
5 cl, 14 dwg, 11 ex