RECOMBINANT PLASMID DNA pET-GST-6His-GM, PROVIDING EXPRESSION AND SYNTHESIS IN THE PROKARYOTIC SYSTEM OF E. COLI OF THE RECOMBINANT HYBRID PROTEIN GST-6His-GM, THE RECOMBINANT STRAIN OF ESCHERICHIA COLI BL21 (DE3) / pET-GST-6His-GM AND THE RECOMBINANT PROTEIN OF HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF), WHICH HAS HEMOSTIMULATING ACTIVITY

Abstract

FIELD: biotechnology.SUBSTANCE: recombinant plasmid DNA pET-GST-6His-GM is proposed, which provides expression in the prokaryotic system of E. coli of the recombinant hybrid protein GST-6His-GM, having the amino acid sequence SEQ ID NO: 2. The said recombinant plasmid DNA has a size of 6425 bp and a nucleotide sequence of SEQ ID NO: 1. Also provided is a recombinant Escherichia coli strain BL21(DE3) / pET-GST-6His-GM, obtained by transforming the Escherichia coli strain BL21(DE3) with the said recombinant plasmid DNA pET-GST-6His-GM and producing a recombinant hybrid protein GST-6His-GM having the amino acid sequence SEQ ID NO: 2, used to produce recombinant human GM-CSF protein.EFFECT: consistently higher yield of recombinant human GM-CSF protein and simplifies its subsequent purification.2 cl, 6 dwg, 2 tbl, 5 ex

Description

[0001] The invention relates to recombinant plasmid DNA pET-GST-6His-GM, which provides expression and synthesis in the prokaryotic system of E. coli of the recombinant chimeric protein GST-6His-GM, the recombinant strain of Escherichia coli BL21(DE3) / pET-GST-6His-GM and the recombinant protein of human granulocyte-macrophage colony-stimulating factor (GM-CSF), which has hemostimulating activity and can be used in genetic engineering, biotechnology and medicine.

[0002] Recombinant GM-CSF protein can serve as a basis for the creation of therapeutic and immunobiological drugs.

[0003] Technology Level

[0004] GM-CSF is a natural cytokine protein that regulates the proliferation and differentiation of stem cells of granulocyte and monocyte / macrophage progenitor cells, and also ensures the activation of mature neutrophils, monocytes and eosinophils [Zurochka AV, Zurochka VA, Dobrynina MA, Gritsenko VA Immunobiological properties of granulocyte-macrophage colony-stimulating factor and synthetic peptides of its active center / / Medical Immunology, 2021. Vol. 23, No. 5. Pp. 1031-1054. [Zurochka AV, Zurochka VA, Dobrynina MA, Gritsenko VA Immunobiological properties of granulocyte-macrophage colony-stimulating factor and synthetic peptides of its active center. Meditsinskaya immunologiya = Medical Immunology (Russia), 2021, Vol. 23, no. 5, pp. 1031-1054. (In Russ.)] doi: 10.15789 / 1563-0625-IPO-2216.].Human GM-CSF, a 144-amino acid peptide, is an FDA-approved cytokine used to treat immunodeficiency in cases of neutropenia, acute myeloid leukemia, myeloid reconstitution, and bone marrow transplantation [Vanitha Selvarajan, Anil Parsram Bidkar, Rajib Shome, Aditi Banerjee, Nidhi Chaubey, Siddhartha S. Ghosh, Pallab Sanpui, Studying in vitro phagocytosis of apoptotic cancer cells by recombinant GMCSF-treated RAW 264.7 macrophages, International Journal of Biological Macromolecules, Volume 102, 2017, Pages 1138–1145, ISSN 0141-8130, https: / / doi.Org / 10.1016 / j.ijbiomac.2017.05.003.].

[0005] When constructing new producer strains, one of the most popular approaches is the expression of a recombinant protein as a chimeric polypeptide, when the target protein is expressed fused with an additional protein. In such cases, the most frequently used proteins that enhance the expression level of the expressed protein and also facilitate the purification of the target protein are maltose-binding protein, glutathione-8-transferase, and thioredoxin [Khairullin R.F. Expression of recombinant proteins in E. coli: a tutorial / R.F. Khairullin, R.G. Kiyamova, A.A. Rizvanov. Kazan: Kazan University Publishing House, 2018. 142 p.]. Closest analogues.

[0006] Recombinant plasmid DNA p280 GM is known, encoding a polypeptide with the properties of human granulocyte-macrophage colony-stimulating factor, and the Escherichia coli strain SG20050 / p280_GM is a producer of a polypeptide with the properties of human granulocyte-macrophage colony-stimulating factor (patent for invention RU No. 2091488, IPC C12N 15 / 70, published on September 27, 1997). The E. coli strain SG 20050 / p280GM contains, as part of a recombinant plasmid, a synthetic gene for granulocyte-macrophage colony-stimulating factor, which determines the constitutive synthesis of a polypeptide with the properties of human GM-CSF, and ensures a GM-CSF expression level of approximately 15% of the total cellular protein.

[0007] The disadvantage of this analogue is the uncontrolled expression of the GM-CSF gene and the low yield of the desired protein, the isolation of which in preparative quantities will require the expansion of producers in large volumes.

[0008] The closest analogue (prototype) is the recombinant plasmid DNA p280_2GM, encoding a polypeptide with the properties of human granulocyte-macrophage colony-stimulating factor, the E. coli strain SG 20050 / p280_2GM - a producer of a polypeptide with the properties of human granulocyte-macrophage colony-stimulating factor and a method for producing the said polypeptide (Patent for Invention RU No. 2708556, IPC C12N 15 / 63, published on 09.12.2019). The E. coli strain SG 20050 / p280_2GM contains a 387 bp DNA fragment in the recombinant plasmid p280_2GM, which encodes the amino acid sequence of mature human GM-CSF; has a molecular weight of 2.95 MDa (4422 bp); consists of: PsTI / BamHI(pol)-fragment of DNA of plasmid p280GM (1484 bp), containing the C-terminal part of the beta-lactamase gene, tandem promoters of the E. coli tryptophan operon and the synthetic human GM-CSF gene, as well as EcoRI(pol) / PstI (2938 bp).) - a fragment of the same plasmid containing the synthetic human GM-CSF gene and the N-terminal portion of the beta-lactamase gene; contains: - a tandem of promoters of the tryptophan operon E. The prototype method allows for a level of human GM-CSF synthesis of approximately 25 - 30% of the total protein at a density of 10. 9 cells / ml, and a yield of up to 17 mg of highly purified target protein from 1 liter of culture fluid, which is 1.5 - 2 times higher than in the analogous method according to Russian patent No. 2091488.

[0009] The disadvantage of the invention is the constitutive synthesis of the target product, which does not guarantee its consistently high yield in biomass.

[0010] Disclosure of invention

[0011] The technical result of the claimed invention is to ensure a consistently higher yield of human granulocyte-macrophage colony-stimulating factor and to simplify its subsequent purification by cloning the nucleotide sequence of GST-6His-GM as part of the pET21 plasmid vector, which provides expression of foreign genes under the control of a powerful inducible T7 promoter, and creating the Escherichia coli BL 21(DE3) / pET-GST-6His-GM strain, a superproducer of the recombinant chimeric protein GST-6His-GM, for isolating the desired product in preparative quantities from minimal volumes of biomass.

[0012] The technical result is achieved by creating a recombinant plasmid DNA pET-GST-6His-GM, which ensures expression in the prokaryotic system of E. coli of the recombinant chimeric protein GST-6His-GM, having the nucleotide sequence SEQ ID NO: 1, a size of 6425 bp and containing, in accordance with the physical and genetic map presented in Fig. 1, the following elements:

[0013] ori - the replication origin site ori, with coordinates from 1630 to 2218 bp; lacI - the sequence encoding the lactose operon repressor and with coordinates from 3648 to 4730 bp;

[0014] lacI promoter - a sequence encoding the lacI promoter and having coordinates from 4731 to 4808 bp;

[0015] T7 promoter - a sequence encoding the promoter of bacteriophage T7 and having coordinates from 5117 to 1057 bp;

[0016] lac operator - a sequence encoding the lactose operon operator, which controls gene expression and has coordinates from 5136 to 5160 bp; RBS - a sequence encoding the ribosome entry site and has coordinates from 5191 to 5196 bp;

[0017] GST is a sequence encoding glutathione-S-transferase and has coordinates from 5205 to 5858 bp;

[0018] 6His is a sequence encoding a polyhistidine tag for purification of the recombinant protein using metal chelate chromatography and has coordinates from 5859 to 5876 bp;

[0019] TEV - a sequence encoding a region hydrolyzed by TEV protease and having coordinates from 5877 to 5897 bp;

[0020] GM is a sequence encoding mature human granulocyte-macrophage colony-stimulating factor and has coordinates from 5898 to 6278 bp;

[0021] T7 terminator - the sequence of the transcription terminator of bacteriophage T7, having coordinates from 6353 to 6400 bp;

[0022] AmpR promoter - bacterial promoter of the ampicillin resistance gene, having coordinates from 3684 to 3788 bp;

[0023] AmpR is a gene for resistance to the antibiotic ampicillin, which allows for plasmid amplification in E. coli and has coordinates from 3789 to 4649 bp.

[0024] The technical result is also achieved by creating a stable recombinant strain of Escherichia coli BL21(DE3) / pET-GST-6His-GM, obtained by transforming the strain of Escherichia coli BL21(DE3) with the recombinant plasmid DNA pET-GST-6His-GM according to claim 1 and producing a recombinant chimeric protein GST-6His-GM, having a molecular weight of 41.6 kDa and an amino acid sequence of SEQ ID NO: 2. The final yield of the chimeric protein biomass averaged 3 - 3.5 g per 1 l of culture fluid, and the final yield of the target protein was 20 mg per 1 l of culture fluid.

[0025] The specified technical result is also achieved by obtaining a recombinant human GM-CSF protein that has hemostimulating activity, has the amino acid sequence SEQ ID NO: 3, a molecular weight of 14.6 kDa, obtained from a recombinant chimeric protein GST-6His-GM produced by the recombinant Escherichia coli strain BL21(DE3) / pET-GST-6His-GM according to claim 2, containing the recombinant plasmid DNA pET-GST-6His-GM according to claim 1, by cleaving the chimeric protein with TEV protease (TEVp) and purifying the target protein from TEVp and GST by affinity chromatography on a column with IMAC Sepharose.

[0026] The invention is explained by the graphic materials presented in Figs. 1-6. Fig. 1 shows the nucleotide sequence (SEQ ID NO: 1) of the plasmid genetic construct pET-GST-6His-GM. Fig. 2 shows the amino acid sequence (SEQ ID NO: 2) of the recombinant chimeric protein GST-6His-GM. Fig. 3 shows the amino acid sequence (SEQ ID NO: 3) of the recombinant protein rhGM-CSF. Fig. 4 shows the physical and genetic map of the plasmid vector pET-GST-6His-GM, including the nucleotide sequence of the recombinant chimeric protein GST-6His-GM; fl (+) ori - the origin of single-stranded DNA replication; AmpR is the ampicillin resistance gene, ori is the origin of replication; lad is the sequence encoding the lactose operon repressor; 6His is a polyhistidine tag for purification of the recombinant protein using metal chelate chromatography. Fig. 5 shows the electropherogram and densitogram of cell lysates of the E. coli biomass.coli BL21 / pET-GST-6His-GM before and after IPTG induction. Electrophoresis in 12% PAGE, Coomassie R-250 staining. Lanes: 1 - protein molecular weight marker (10-250 kDa); 2 - cell lysate of E. coli BL21 / pET-GST-6His-GM biomass before induction; 3 - cell lysate of E. coli BL21 / pET-GST-6His-GM biomass after 6 h of IPTG induction. Fig. 6 shows an electropherogram of the rhGM-CSF protein purification products, 15%) PAGE, non-reducing conditions, Coomassie R-250 staining. Lanes: 1 - protein molecular weight marker (10-250 kDa); 2 - fusion protein after affinity chromatography on Glutathione Resin; 3 - protein mixture after TEVp hydrolysis; 4 - rhGM-CSF protein substance (final product).

[0027] For a better understanding of the essence of the proposed invention, examples of its implementation are given below. All standard genetic engineering and microbiological manipulations, as well as DNA amplification and sequencing were carried out according to known methods [Maniatis T., Fritsch E., Sambrook J. Molecular Cloning, Moscow: Mir, 1984; DNA Cloning. Methods. Ed. D. Glover, Trans. from English, Moscow, Mir, 1988; Saiki RK et al. Science. 1988, 239(4839):487-491; Sanger F. et al. Proc. Nat. Acad. Sci. 1977, 74:5463-5467]. Example 1. Construction of the pET-GST-6His-GM plasmid construct, which ensures the synthesis of the recombinant GST-6His-GM protein. The sequence of the gene encoding granulocyte-macrophage colony-stimulating factor was cloned into the pET21 vector. Amplification of the GST and GM nucleotide sequences was performed using primer pairs GM-GST-F, GM-GST-R and TEV-GM-F, MG-R, respectively (Table 1).For GST-6His-GM, the pGH-GM plasmid, containing the synthetic GM-CSF gene, was used as a template; for GST, the pGEX-4T-1 plasmid was used. The PCR products were then mixed, and overlapping amplification was performed using flanking primers. The resulting PCR product and the pET21a(-) vector were treated with FauNDI and CciNI restriction enzymes (SibEnzyme LLC, Russia). The hydrolysis products were then mixed and ligated using bacteriophage T4 DNA ligase (SibEnzyme LLC, Russia) for 30 minutes at room temperature. The resulting ligation mixture was used to transform competent E. coli cells of the Neb Stable strain.

[0028] A primary test for the presence of the insert was performed using colony-based PCR. The amplification products were separated on a 1% agarose gel followed by staining with ethidium bromide (0.5 μg / ml).

[0029] Positive colonies were cultured in 5 ml of LB medium with ampicillin (50 μg / ml) overnight at 37°C at 170 rpm. Then, plasmid DNA was isolated from bacterial cells using commercial DNA mini kits from Qiagen according to the manufacturer's recommendations. The primary structure of the expression vector was confirmed by sequencing. Sequencing was performed using the Sanger method at the Genomics Collective Use Center of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the recombinant plasmid pET-GST-6His-GM with the nucleotide sequence SEQ ID NO: 1 (Fig. 1) was obtained, providing the synthesis of the chimeric protein GST-6His-GM.

[0030] Sequencing the plasmid DNA of positive clones in the insertion region allowed us to select clones free of insertion gene defects (insertions, deletions, substitutions). Target plasmid DNA was then prepared and isolated from these clones. The physical and genetic map is shown in Fig. 4.

[0031]

[0032] Example 2. Obtaining an E. coli strain producing a recombinant protein.

[0033] When creating a prokaryotic expression system for the GST-6His-GM gene encoding granulocyte-macrophage colony-stimulating factor, the BL21(DE3) strain of E. coli cell culture (Novagen, USA) was used.

[0034] 2.1. Transformation of "competent" E. coli cells with a recombinant plasmid. 10 μl of the ligation mixture were added to "competent" E. coli BL21(DE3) cells, and the cells were incubated on ice for 30 minutes. After this, the cells were subjected to a "temperature shock" at 42°C for 45 seconds. The cells were cooled on ice for 2 minutes, then 200 μl of the "SOB" (Super Optimal Broth) medium were added and the cells were incubated at 37°C for 60 minutes. After incubation, the transformed cells were plated on a Petri dish with a solid nutrient medium LB (Lysogeny broth) (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml).

[0035] 2.2. Cultivation of transformed E. coli cell culture and induction of recombinant GST-6His-GM protein synthesis

[0036] E. coli strain BL21(DE3) cells transformed with the pET-GST-6His-GM vector were selectively cultured in 100 ml of liquid LB nutrient medium supplemented with ampicillin sodium at a working concentration of 20 μg / ml. Synthesis of the target recombinant protein was induced with 0.5 mM IPTG (isopropyl-β-D-thiogalactoside). The selection of E. coli producer clones was carried out based on the presence of the synthesized target protein based on the results of electrophoresis of cell lysates in 10% polyacrylamide gel (Fig. 5) with sodium dodecyl sulfate (SDS-PAGE) (see Example 3). As a control, a non-induced lysate of E. coli cells of the BL21(DE3) strain, obtained in a similar manner, containing the vector plasmid pET-GST-6His-GM, was used.

[0037] Example 3. Protein electrophoresis in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Electrophoresis was performed in a resolving gel (composition: 30% acrylamide; 1.5 M Tris (pH 8.8); 10% SDS; 10% ammonium persulfate; TEMED (tetramethylethylenediamine) 1 μl / ml) and a stacking gel (composition: 30% acrylamide; 1 M Tris (pH 6.8); 10% SDS; 10% ammonium persulfate; TEMED 1 μl / ml) in different buffer solutions (upper - 5X Tris-glycine buffer solution pH 8.3 (composition: 1.25 mM Tris; 1.25 M glycine; 0.5% SDS), lower - 1X Tris-acetate buffer solution pH 8.0 (composition: 40 mM Tris - HCl; 40 mM acetic acids; 2 mM EDTA (ethylenediaminetetraacetic acid). The biological material was mixed with the loading buffer, boiled for 5 minutes, and loaded onto the upper stacking gel. SDS-PAGE was run at a voltage of ~10 V / cm in the stacking gel and -180 V in the resolving gel. The gels were stained with Coomassie R-250 solution.

[0038] Example 4. Purification of recombinant rhGM-CSF protein under denaturing conditions. The biomass sample (10 g of wet cells) was suspended in 100 ml of 20 mM Tris-HCl, pH 8.0, buffer containing 1 mM phenylmethylsulfonyl fluoride, a protease inhibitor, and destroyed by ultrasound on a Sonicator Q2000 (Qsonica, USA) until the optical density at 550 nm (OD595) decreased by 50% of the initial value, followed by centrifugation. Inclusion bodies (IB) were separated by centrifugation on an Avanti J-30I centrifuge (Beckman Coulter, USA) at 12,000 min -1, (4±2)°C, 30 min. The TB was washed with 150 ml of 20 mM Tris-HCl, pH 8.0, containing 5 mM EDTA or 0.5% Triton X-100. Protein denaturation was performed in 100 ml of 8 M urea in 20 mM Tris, pH 8.0. Renaturation was by diluting 10 times to 1 l of 20 mM Tris, pH 8.0. Purification of the fusion protein was carried out by affinity chromatography on a column with Glutathione Seplife 4FF, (SunResin), 10 ml volume, equilibrated with PBS, pH 7.6. The fusion protein was eluted with a solution containing 10 mM glutathione in PBS, pH 7.6. The fusion protein was digested with TEV protease (TEVp) (Biolabmix LLC). Affinity chromatography on an 8 ml IMAC-Sepharose column (Cytiva, Sweden) equilibrated with 20 mM imidazole, 20 mM Tris-HCl, pH 8.0 was used to purify the target protein from TEVp and GST. The target protein was not adsorbed and remained in the wash buffer.Analysis of the target protein (14.6 kDa) in the chromatographic fractions was performed by electrophoresis in 15% polyacrylamide gel under non-reducing conditions with Coomassie R-250 staining (Fig. 6). Fractions containing the target protein were pooled and sterilized using a 0.22 μm bacteriological filter (Corning, USA) and stored at -20°C. The yield of the target protein was 20 mg ± 0.5 mg of target protein from 1 L of culture fluid, purity greater than 95%. Example 5. Evaluation of specific (granulocyte-stimulating) activity. Hemostimulating activity was assessed using a model of cytostatic myelosuppression induced by the administration of cyclophosphamide (CPh) to mice. We used 18 male CBA mice, weighing 20-24 g (2.5-3.0 months old), obtained from the nursery of the Federal State Budgetary Scientific Institution of Virology and Biotechnology "Vector" of Rospotrebnadzor (Koltsovo, Novosibirsk Region).

[0039] To reproduce the model, all animals received a single intraperitoneal injection of cyclophosphamide at a dose of 200 mg / kg. A day later, the animals of the experimental groups began a course of subcutaneous injections of the drugs. The first experimental group was administered a control sample of the rhGM-CSF substance, the second - rhGM-CSF substance lot 010424 at a dose of 90 μg / kg in a volume of 0.2 ml per 20 g of body weight once a day for 4 days. This dose was determined to be effective in previous experiments on mice. Mice in the control group received sodium chloride injection solution 0.9% according to the same scheme as the experimental ones. Each group consisted of 6 mice. The intact group did not receive any injection.

[0040] On the fifth day, blood samples were collected from the animals' tail tips for analysis. The total leukocyte count was determined in the blood samples, and the white blood cell count was calculated [Novitsky V.V., Evtushenko O.M. Guide to Practical Classes in Hematology. - Tomsk, 1999]. Table 2 presents the results of the hemostimulating activity assessment in the cytostatic myelosuppression model.

[0041]

[0042] The level of hemostimulating activity of the drugs was calculated as the ratio of the absolute number of segmented neutrophils in the blood of experimental animals to the absolute number of segmented neutrophils in the control animals (in percent).

[0043] rhGM-CSF at a dose of 90 μg / kg caused a 235% increase in the absolute content of segmented neutrophils compared to the control value (Table 2). Table 2 presents the results of the hemostimulating activity assessment in the cytostatic myelosuppression model.

[0044] Thus, the claimed technical solution enables the production of the pET-GST-6His-GM genetic construct, containing the gene for the recombinant chimeric protein GST-6His-GM. The E. coli BL21 / DE3(+) strain, transformed with the recombinant plasmid and induced by IPTG, synthesizes the recombinant chimeric protein GST-6His-GM with a molecular weight of 41.6 kDa. The recombinant GST-6His-GM protein contains a polyhistidine tract for affinity purification and a TEV proteolysis site for producing rhGM-CSF. This approach enables the production of highly purified recombinant GM-CSF. The final yield of the chimeric protein biomass averaged 3–3.5 g per 1 liter of culture fluid, and the final yield of the target protein was 20 mg per 1 liter of culture fluid, which confirms the stated technical result.

[0045] --->

[0046] <?xml version="1.0" encoding="UTF-8"?>

[0047] <!DOCTYPE ST26SequenceListing PUBLIC "- / / WIPO / / DTD Sequence Listing

[0048] 1.3 / / EN" "ST26SequenceListing_V1_3.dtd">

[0049] <ST26SequenceListing dtdVersion="V1_3" fileName="Приложение_ДНК

[0050] pET-GST-6His-GM, рекомбинантный штамм и ГМ-КСФ, .xml"

[0051] softwareName="WIPO Sequence" softwareVersion="2.3.0"

[0052] productionDate="2024-10-20">

[0053] <applicationidentification>

[0054] <ipofficecode>RU< / ipofficecode>

[0055] <applicationnumbertext> 123456< / applicationnumbertext>

[0056] <filingdate> 2024-10-16< / filingdate>

[0057] < / applicationidentification>

[0058] <applicantfilereference> 123< / applicantfilereference>

[0059] <earliestpriorityapplicationidentification>

[0060] <ipofficecode> RU< / ipofficecode>

[0061] <applicationnumbertext> 1234567< / applicationnumbertext>

[0062] <filingdate> 2024-10-09< / filingdate>

[0063] < / earliestpriorityapplicationidentification>

[0064] <applicantname languagecode="ru">Federal budgetary institution

[0065] Science "State Research Center of Virology and Biotechnology

[0066] Vector of the Federal Service for Supervision of Consumer Rights Protection

[0067] consumers and human well-being (State Research Center of Virology and Biotechnology "Vector"

[0068] Rospotrebnadzor)< / applicantname>

[0069] <applicantnamelatin>Federal budgetary scientific institution

[0070] State Research Center of Virology and Biotechnology

[0071] Vector of the Federal Service for Supervision in the Sphere of Defense

[0072] Consumer Rights and Human Welfare (FBUN GNTS VB

[0073] "Vector" Rospotrebnadzor) (RU)< / applicantnamelatin>

[0074] <inventiontitle languagecode="ru">Recombinant plasmid DNA

[0075] pET-GST-6His-GM, providing expression and synthesis in

[0076] prokaryotic system E.coli recombinant chimeric protein

[0077] GST-6His-GM, recombinant Escherichia coli strain

[0078] BL21(DE3) / pET-GST-6His-GM and recombinant protein

[0079] Human granulocyte-macrophage colony-stimulating factor

[0080] (GM-CSF), which has hemostimulating activity< / inventiontitle>

[0081] <sequencetotalquantity> 3< / sequencetotalquantity>

[0082] <sequencedata sequenceidnumber="1">

[0083] <insdseq>

[0084] <INSDSeq_length>6425< / INSDSeq_length>

[0085] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0086] <INSDSeq_division>PAT< / INSDSeq_division>

[0087] <INSDSeq_feature-table>

[0088] <insdfeature>

[0089] <INSDFeature_key>source< / INSDFeature_key>

[0090] <INSDFeature_location>1..6425< / INSDFeature_location>

[0091] <INSDFeature_quals>

[0092] <insdqualifier>

[0093] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0094] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0095] < / insdqualifier>

[0096] <insdqualifier id="q2">

[0097] <INSDQualifier_name>organism< / INSDQualifier_name>

[0098] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0099] < / insdqualifier>

[0100] < / INSDFeature_quals>

[0101] < / insdfeature>

[0102] < / INSDSeq_feature-table>

[0103] <INSDSeq_sequence>tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtg

[0104] gtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccctt

[0105] cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatt

[0106] tagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccc

[0107] tgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactg

[0108] gaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattg

[0109] gttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttca

[0110] ggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgt

[0111] atccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattca

[0112] acatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacg

[0113] ctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaaca

[0114] gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct

[0115] atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcag

[0116] aatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattat

[0117] gcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaa

[0118] ggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctg

[0119] aatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaac

[0120] tattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagt

[0121] tgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgag

[0122] cgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctaca

[0123] cgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaa

[0124] gcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattt

[0125] aaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttcc

[0126] actgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctg

[0127] ctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctt

[0128] tttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttag

[0129] gccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgc

[0130] tgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcgg

[0131] tcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc

[0132] tacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcgg

[0133] cagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtc

[0134] gggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaa

[0135] acgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgc

[0136] gttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga

[0137] acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgc

[0138] atctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagtta

[0139] agccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgc

[0140] tgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagc

[0141] tgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgt

[0142] ggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgt

[0143] taatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctcc

[0144] gtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacggg

[0145] ttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcg

[0146] ggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggt

[0147] agccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagac

[0148] tttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtc

[0149] gcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgg

[0150] gtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttct

[0151] cgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgc

[0152] aagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgcc

[0153] ggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcg

[0154] cccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagt

[0155] gagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctg

[0156] cattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttc

[0157] accagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtcca

[0158] cgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtc

[0159] ttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgc

[0160] attgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcattt

[0161] gcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgatt

[0162] gcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaac

[0163] agcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggaga

[0164] aaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagc

[0165] ttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgaga

[0166] agattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcac

[0167] ccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggt

[0168] ggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagc

[0169] tccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcggg

[0170] aaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccac

[0171] cctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtcc

[0172] gggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttg

[0173] agcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctg

[0174] ccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgat

[0175] gtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtag

[0176] aggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattc

[0177] ccctctagaaataattttgtttaactttaagaaggagatatacatatgtcccctatactaggttattgga

[0178] aaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcattt

[0179] gtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatctt

[0180] ccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagc

[0181] acaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatat

[0182] tagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaag

[0183] ctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaa

[0184] cccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgc

[0185] gttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatcc

[0186] agcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaac

[0187] atcatcaccatcaccatgaaaatctttattttcaaggtgcaccggcacgtagcccgagtccgagcacaca

[0188] gccgtgggaacatgttaatgcaattcaagaagcacgtcgtctgctgaatctgagccgtgataccgcagca

[0189] gaaatgaatgaaaccgttgaagttatcagcgagatgtttgatctgcaagaaccgacctgcctgcagaccc

[0190] gtctggaactgtataaacagggtctgcgtggtagcctgaccaaactgaaaggtccgctgaccatgatggc

[0191] aagccattataaacagcattgtccgcctacaccggaaaccagctgtgcaacccagattattacctttgag

[0192] agctttaaagagaacctgaaagatttcctgctggtgattccgtttgattgttgggaacctgttcaagaat

[0193] aagcggccgcgatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaa

[0194] taactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatat

[0195] ccggat< / INSDSeq_sequence>

[0196] < / insdseq>

[0197] < / sequencedata>

[0198] <sequencedata sequenceidnumber="2">

[0199] <insdseq>

[0200] <INSDSeq_length>358< / INSDSeq_length>

[0201] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0202] <INSDSeq_division>PAT< / INSDSeq_division>

[0203] <INSDSeq_feature-table>

[0204] <insdfeature>

[0205] <INSDFeature_key>source< / INSDFeature_key>

[0206] <INSDFeature_location>1..358< / INSDFeature_location>

[0207] <INSDFeature_quals>

[0208] <insdqualifier>

[0209] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0210] <INSDQualifier_value>protein< / INSDQualifier_value>

[0211] < / insdqualifier>

[0212] <insdqualifier id="q4">

[0213] <INSDQualifier_name>organism< / INSDQualifier_name>

[0214] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0215] < / insdqualifier>

[0216] < / INSDFeature_quals>

[0217] < / insdfeature>

[0218] < / INSDSeq_feature-table>

[0219] <INSDSeq_sequence>MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELG

[0220] LEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK

[0221] VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI

[0222] DKYLKSSKYIAWPLQGWQATFGGGDHPPKHHHHHHENLYFQGAPARSPSPSTQPWEHVNAIQEARRLLNL

[0223] SRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCAT

[0224] QIITFESFKENLKDFLLVIPFDCWEPVQE< / INSDSeq_sequence>

[0225] < / insdseq>

[0226] < / sequencedata>

[0227] <sequencedata sequenceidnumber="3">

[0228] <insdseq>

[0229] <INSDSeq_length>128< / INSDSeq_length>

[0230] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0231] <INSDSeq_division>PAT< / INSDSeq_division>

[0232] <INSDSeq_feature-table>

[0233] <insdfeature>

[0234] <INSDFeature_key>source< / INSDFeature_key>

[0235] <INSDFeature_location>1..128< / INSDFeature_location>

[0236] <INSDFeature_quals>

[0237] <insdqualifier>

[0238] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0239] <INSDQualifier_value>protein< / INSDQualifier_value>

[0240] < / insdqualifier>

[0241] <insdqualifier id="q6">

[0242] <INSDQualifier_name>organism< / INSDQualifier_name>

[0243] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0244] < / insdqualifier>

[0245] < / INSDFeature_quals>

[0246] < / insdfeature>

[0247] < / INSDSeq_feature-table>

[0248] <INSDSeq_sequence>GAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFD

[0249] LQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIP

[0250] FDCWEPVQE< / INSDSeq_sequence>

[0251] < / insdseq>

[0252] < / sequencedata>

[0253]

[0254] <---

Claims

1. Recombinant plasmid DNA pET-GST-6His-GM, providing expression in the prokaryotic system of E. coli of the recombinant chimeric protein GST-6His-GM, having the amino acid sequence of SEQ ID NO: 2, used to obtain the recombinant protein of human granulocyte-macrophage colony-stimulating factor (GM-CSF), possessing hemostimulating activity, having the nucleotide sequence of SEQ ID NO: 1, a size of 6425 bp and containing the following elements: - ori - the site of the origin of replication ori, with coordinates from 1630 to 2218 bp; - lacl is a sequence encoding the lactose operon repressor and having coordinates from 3648 to 4730 bp; - lacl promoter - a sequence encoding the lacl promoter and having coordinates from 4731 to 4808 bp; - T7 promoter - a sequence encoding the promoter of bacteriophage T7 and having coordinates from 5117 to 1057 bp; - lac operator - a sequence encoding the lactose operon operator, which controls gene expression and has coordinates from 5136 to 5160 bp; - RBS is a sequence encoding the ribosome landing site and has coordinates from 5191 to 5196 bp; - GST is a sequence encoding glutathione-S-transferase and has coordinates from 5205 to 5858 bp; - 6His is a sequence encoding a polyhistidine tag for purification of the recombinant protein using metal chelate chromatography and has coordinates from 5859 to 5876 bp; - TEV is a sequence encoding a region hydrolyzed by TEV protease and having coordinates from 5877 to 5897 bp; - GM is a sequence encoding mature human granulocyte-macrophage colony-stimulating factor and has coordinates from 5898 to 6278 bp; - T7 terminator - the sequence of the transcription terminator of bacteriophage T7, having coordinates from 6353 to 6400 bp; - AmpR promoter - bacterial promoter of the ampicillin resistance gene, having coordinates from 3684 to 3788 bp; - AmpR is a gene for resistance to the antibiotic ampicillin, which allows for plasmid amplification in E. coli and has coordinates from 3789 to 4649 bp.

2. Recombinant Escherichia coli strain BL21(DE3) / pET-GST-6His-GM, obtained by transforming the Escherichia coli strain BL21(DE3) with the recombinant plasmid DNA pET-GST-6His-GM according to claim 1 and producing a recombinant chimeric protein GST-6His-GM, having the amino acid sequence of SEQ ID NO: 2 and used to obtain recombinant human GM-CSF protein, possessing hemostimulating activity and having the amino acid sequence of SEQ ID NO: 3.

Images