RU2744592C1 - Recombinant plasmid pet21-tf7, providing synthesis of modified tissue factor, and escherichia coli bl21 (de3) pet21-tf7 strain - producer of recombinant human tissue factor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions refers to recombinant plasmid providing synthesis of recombinant modified human tissue factor, and recombinant strain producing human recombinant tissue factor. Disclosed is a recombinant plasmid pET21-TF7 with size of 6171 base pairs (bp), containing the following structural elements: T7 bacteriophage promoter; recombinant gene coding modified human tissue factor; untranslated region for terminating transcription of the bacterial operon; phage T7 RNA polymerase terminator; sequence bla (AmpR); ColE1 (ori) sequence. Recombinant gene of the modified human tissue factor has a nucleotide sequence SEQ ID NO: 1 nucleotide in position 5205 bp to nucleotide position 5996 bp, which encodes the target protein with the full amino acid sequence SEQ ID NO: 2, built-in FauND I and CciNI restriction sites in the pET21a vector polylinker region, containing a T7 phage TNA RNA polymerase promoter and terminator. Said plasmid provides synthesis of 29.6 kDa recombinant modified human tissue factor in Escherichia coli BL21 (DE3) cells. Also presented is recombinant strain Escherichia coli BL21 (DE3)/pET21-TF7, which produces recombinant human tissue factor and obtained by transformation with said recombinant plasmid pET21-TF7.

EFFECT: group of inventions provides a high and stable yield of the end product.

2 cl, 5 dwg, 1 tbl, 3 ex

Description

The invention relates to a genetic construct (plasmid) that provides the synthesis of a modified protein - human tissue factor (TF), which, after isolation, purification and relipidization, is intended for use as one of the components of a diagnostic product used in the formulation of a clinical laboratory test for determining prothrombin time ( PV) and a recombinant E. coli strain - a producer of modified TF.

Tissue factor is a transmembrane protein that acts as a non-enzymatic cofactor and a high-affinity cellular receptor for coagulation factor (f) VII / VIIa [1-4]. The tissue factor stabilizes the catalytic site of fVIIa on the plasma membrane and ensures its interaction with substrates - fIX and fH. The TF-fVIIa complex is the primary activator of the enzymatic coagulation cascade of the external blood coagulation pathway [5, 6]. Normally, TF is not expressed on the surface of cells that come into contact with circulating blood [7]. In case of damage to the blood vessel wall caused by trauma or inflammation, subendothelial TF is exposed to the bloodstream and binds to factor VII / VIIa, which circulates as an enzyme with minimal (trace) proteolytic activity [8-11]. As part of the TF-fVII / VIIa complex, the catalytic efficiency of fVIIa increases by several orders of magnitude and initiates blood coagulation by proteolytic activation of zymogens of factors IX and X and their conversion into active serine proteinases -phIXa and fXa [12].

The human TF gene is located on chromosome 1 (1p22-p21), has 12,640 nucleotides and consists of 6 exons. The open reading frame of the TF gene encodes a protein with a molecular weight of 33 kDa, consisting of 295 amino acids, including a signal sequence of 32 amino acid residues (a.a.). Three domains are distinguished in the TF structure: extracellular (a.o. 1-219), transmembrane (a.o. 220-242) and cytoplasmic (a.o. 243-263) [UniProtKB - PI3726 (TF_HUMAN) - https: / /www.uniprot.org/uniprot/P13726].

The spatial structure of the extracellular region is represented by two Ig-like domains [13, 14]. The extracellular domain of TF interacts with fVII / VIIa to form a highly active complex enzyme. In this case, the maximum catalytic efficiency of the TF-fVII / VIIa complex is manifested only if it is bound to a phospholipid membrane of a certain composition [15-17]. It is now generally accepted that, as a coagulation initiator, TF without a cytoplasmic domain is functionally identical to a full-length protein. According to Fiore et al. [18] recombinant TF (rTF), devoid of both the cytoplasmic and transmembrane domains, is unable to bind to the phospholipid membrane, and the resulting complex of the extracellular domain with fVII exhibits very low procoagulant activity. Thus, two of the three TF domains - extracellular and transmembrane - play an important role in the blood coagulation process, while the cytoplasmic region is associated with signaling functions [19].

In combination with phospholipids, TF forms tissue thromboplastin (TP), one of the hemostatic factors involved in the external pathway of blood coagulation. In clinical practice, TP preparations are widely used in the formulation of a PT determination test, for detecting disorders in the blood coagulation system, and for monitoring treatment with indirect anticoagulants [1, 5, 15, 20]. The development of new highly active, standardized and sensitive TP preparations is an urgent task of biotechnology, since it is associated with the prospect of increasing the sensitivity and reproducibility of clinical laboratory studies of the human hemostasis system.

Traditionally, to obtain natural TP preparations, the human placenta, lung tissue and the brain of a cow or rabbit are used. The disadvantages of these drugs include the possibility of contamination with activated blood coagulation factors and other contaminants that can affect the interpretation of the results. Another serious problem is the standardization of reagents for clinical diagnostics obtained from various types of biological raw materials. To standardize natural reagents for the determination of PO, WHO has introduced a system for calculating the International Normalized Ratio (MHO) [21]. The MHO adjusts the PT values obtained using TP preparations of various origins by comparison with the international reference TP preparation, which is assigned an international susceptibility index (MIC). The index is a measure of the sensitivity of a given TP drug, in relation to the international reference drug. MHO is calculated as the ratio of the patient's PT to the normal PT value, raised in MFI:

MHO = (MF patients / normal value PX) ^ISI

Since 1983, manufacturers of thromboplastins for clinical laboratory diagnostics are required to determine and indicate the MIC for each batch of TR. For highly sensitive drugs, the MIC value approaches 1.0.

Standardization is not the only problem associated with the production and use of natural TR products. Thromboplastins made from human tissue can be sources of HIV-1 and other dangerous viral infections. In addition, TP preparations of animal origin differ in their sensitivity to coagulation factor deficiency and proteins synthesized under conditions of vitamin K deficiency or in the presence of its antagonists. The availability of highly purified TF preparations from natural sources has been limited in recent years due to their high cost [20, 22, 23].

A logical solution aimed at eliminating these disadvantages is the production of diagnostic TP preparations using genetically engineered human TF and artificial liposomes optimized for phospholipid composition [15, 24, 25]. The main advantage of recombinant thromboplastin (rTP) preparations produced using human rTF and a complex of synthetic phospholipids is that they do not contain impurities of other coagulation factors. This provides a high sensitivity of rTP preparations to the deficiency of factors of the external blood coagulation pathway [20].

Full-length human rTP variants were synthesized in the expression systems of Escherichia coli [26], Baculovirus [27], and in kidney cells [26]. The extracellular domain of sTF was obtained using genetically modified cells of E. coli [28,2 9], Saccharomyces cerevisiae [29], and Komaga-taella (Pichia) pastoris [30].

Since the free noncellular domain of TF (the so-called “soluble” TF) is unable to activate fVII, it cannot be used to obtain TP preparations that would have sufficient procoagulant activity [31]. Therefore, it is obvious that for the production of highly active TP preparations for clinical diagnostic studies, the TF molecule must contain a transmembrane domain. The presence of a transmembrane domain ensures successful incorporation of recombinant TF (rTF) into artificial liposome preparations [25].

The main disadvantage of existing constructs and producers based on E. coli is the extremely low yield of recombinant TF. This requires the production of a large amount of biomass, significant costs and significantly increases the cost of the product.

In the claimed technical solution, the engineering of the TF structure was carried out, which includes the T7 peptide, which represents the leader sequence (11 amino acids) of the gene 10 of the T7 bacteriophage. The presence of this sequence in the N- or C-terminal part of the protein can significantly increase the yield of the target protein [32].

The technical result of the claimed invention is the creation of a genetic construct "T7 tag - modified human tissue factor", and on its basis - a recombinant strain of Escherichia coli - a producer of recombinant modified human TF, which provides effective synthesis of the target protein.

This technical result is achieved by creating a recombinant plasmid pET21-TF7 with a size of 6171 base pairs (bp), characterized by the physical and genetic map shown in Fig. - 3, providing the synthesis of recombinant human TF of 29.6 kDa in Escherichia coli BL21 (DE3) cells containing the recombinant human TF gene having the nucleotide sequence SEQ ID NO: 1 from the nucleotide at position 5205 bp. to the nucleotide at position 5996 bp, encoding the target recombinant protein with the amino acid sequence SEQ ID NO: 2, inserted at the FauND I and CciNI restriction sites into the polylinker region of the pET21a vector containing the promoter and terminator of T7 RNA polymerase.

The specified technical result is also achieved by creating a recombinant Escherichia coli BL21 (DE3) / pET21-TF7 strain - a producer of recombinant human TF, obtained by transformation with the recombinant plasmid pET21-TF7 according to claim 1.

The claimed strain of Escherichia coli BL21 (DE3) / pET21-TF7 is a producer of recombinant human TF, exhibiting, after inclusion in artificial liposomes, procoagulant activity and providing activation of the external pathway of the hemocoagulation cascade.

To ensure a high and stable yield of the target product, the codon composition of the selected human TF sequence was optimized for expression in E. coli. During the theoretical design, the codons used in eukaryotic cells were replaced with codons used by prokaryotes. The sequence of the leader peptide T7 is added to the nucleotide sequence of the gene at the N-terminus (Fig. 1). It was also decided to remove the cytoplasmic domain from the TF sequence, which does not affect its procoagulant activity. The optimized sequence has been synthesized.

Plasmid pET21a was chosen as the expression vector. After cloning the synthesized sequence in the vector, obtained by the recombinant plasmid, the Neb stable bacterial cells were transformed to produce plasmid DNA.

The E. coli BL21 (DE3) strain was selected as a producer. The choice of the recipient strain is due to the fact that it carries the gene of T7 RNA polymerase, which is necessary to ensure efficient transcription of target genes in the vector construct under the control of the T7 phage promoter, as well as the fact that this strain is defective in protease synthesis, which is essential. increases the yield of synthesized heterologous proteins.

During cultivation, the E. coli BL21 (DE3) / pET21-TF7 strain provides the synthesis of recombinant human TF at the level of 10% of the total bacterial protein.

The invention is illustrated by the following graphical figures. FIG. 1 shows the nucleotide sequence of the recombinant human TF gene. FIG. 2 shows the structure of the amino acid sequence of the recombinant modified human TF. FIG. 3 shows the physical and genetic map of the recombinant plasmid pET21-TF7 containing the sequence of the recombinant modified human TF. FIG. 4 shows a photograph of the electrophoretic separation of the amplification products of the human modified TF gene in a 1% agarose gel. 1 - molecular weight marker, 2, 3, 4 - amplification product of the TF7 sequence. FIG. 5 shows a photograph of the electrophoretic separation of a preparation of recombinant modified human TF in PAGE. 1, 2, 3, 4 - sample of recombinant modified human TF, 5 - molecular weight marker.

For a better understanding of the essence of the invention, examples of its implementation are given below. All standard genetic engineering and microbiological manipulations, as well as DNA amplification and sequencing, were performed according to known techniques.

Example 1. Synthesis, insertion and analysis of a DNA fragment containing the coding sequence of a gene for a recombinant human tissue factor.

The claimed plasmid contains the following structural elements essential for its functioning and expression of the target protein:

- promoter of bacteriophage T7, providing efficient transcription of controlled mRNA; untranslated region of transcription termination of the bacterial operon, terminator of RNA polymerase of phage T7, which provides effective termination of transcription;

- bacterial operon bla (AmpR), encoding beta-lactamase protein, which is a selective marker for transformation of E. coli strains;

- bacterial replication initiation site of the ColE1 (ori) type, providing replication of the plasmid in E. coli strains.

The selected nucleotide sequence of the human TF gene (NP_001984), after optimization of the structure and codon composition, was synthesized on an ABI 3400 DNA / RNA Synthesizer. The synthesized gene was cloned into the pGH cloning vector (DNA synthesis, Moscow). To amplify the TF gene, PCR was performed using oligonucleotide primers containing hydrolysis sites for subsequent insertion of the reaction product into an expression vector: TF7-F (containing a FauNDI site) and TF7-R (containing a CciNI site) (Table 1). The reaction was carried out using PfuSE DNA polymerase (NPO "SibEnzyme", Russia) on a PCR amplifier "BIS" manufactured by OOO "BIS-N" (Russia). Reaction mixture, 50 μl, containing 2 μg DNA (pGH plasmids with tissue factor gene), 10 pM of each primer (67 mM Tris-HCl (pH 8.8), 15 mM ammonium sulfate, 2.5 mM magnesium chloride, 0 , 01% Tween-20, a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP 2.5 mM each) and 1 unit of PfuSE DNA polymerase (NPO SibEnzim, Russia); the reaction was carried out at the following parameters: 50 s - 96 ° С, 30 s - 60 ° С, 1 min - 72 ° С (30 cycles).

After the completion of the amplification, the components of the reaction mixture were separated by electrophoresis in 1% agarose gel, and the reaction product was revealed - a single fragment of 779 bp in size. (Fig 4). The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany). Then, the isolated PCR product and the previously developed and purified vector pET21a were hydrolyzed with restriction endonucleases FauN-DI and CciNI (SibEnzyme, Russia) under the conditions recommended by the manufacturer.

Plasmid DNA after enzymatic hydrolysis was fractionated by EF in 1% agarose gel. The restricted vector was isolated from an agarose gel using a Gel Extraction Kit from Qiagen (Germany). The ligation reaction was performed using bacteriophage T4 DNA ligase (SibEnzyme, Russia). The reaction was carried out at + 4 ° С overnight. The resulting ligase mixture was transformed (as described in example 2) chemically competent cells of E. coli strain NEB (New England Biolabs, UK).

After transformation, clones were selected on selective agar medium LB with the antibiotic ampicillin (50 mg / ml). The presence of the insert of the modified human tissue factor gene in the recombinant plasmid was checked using anilytic PCR. Amplification products were separated in 1% agarose gel followed by staining with ethidium bromide (0.5 μg / ml). Positive colonies were introduced into 5 ml of LB medium with ampicillin (50 μg / ml) and grown overnight on an orbital shaker at 37 ° C and 170 rpm. Then plasmid DNA was isolated from bacterial cells using commercial DNA mini kits from Qiagen (Germany) according to the manufacturer's recommendations. The nucleotide sequence of the obtained plasmids was confirmed by sequencing by the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Russia).

Sequencing of plasmid DNA of positive clones in the insertion region of the modified human TF gene made it possible to select clones without defects in the inserted gene (insertions, deletions, substitutions), after which plasmid DNA was generated and isolated from the selected clones.

Example 2. Obtaining a recombinant Escherichia coli strain BL21 (DE3) / pET21-TF7

In order to obtain a recombinant human TF-producing strain, E. coli BL21 (DE3) cells were transformed with the constructed recombinant plasmid pET21-TF7 (Fig. 3) using calcium chloride.

The cell culture was _grown to OD λ600 = 0.5 optical density units (dp), 1.5 ml of the suspension was centrifuged at 6000 rpm for 2 min in an Eppendorf 5415 R centrifuge. The cell pellet was suspended in 700 μl of 0.2 M CaCl ₂ , incubated at 4 ° C for 15 min, then the cells were pelleted by centrifugation at 4000 rpm for 3 min. The cell pellet was suspended in 100 μl of 0.2 M CaCl ₂ . 50-70 ng of plasmid DNA was added to the cell suspension, incubated for 30 min at 4 ° C, then for 5 min at 37 ° C. 1 ml of LB medium was added to the cell suspension and incubated at 37 ° C for 1 hour. We took 100 μl of culture and plated it on a Petri dish with agar medium supplemented with ampicillin to a concentration of 100 μg / ml. The remainder of the culture was precipitated by centrifugation at 4000 rpm for 3 min, the supernatant was taken, suspended in 100 μl LB, and plated on a plate with agar LB medium containing 100 μg / ml ampicillin. The dishes were incubated in a thermostat at 37 ° C overnight. The next day, individual BL21 (DE3) / pET21-TF7 colonies were identified.

The E. coli BL21 (DE3) strain carrying the recombinant plasmid pET21-TF7, a producer of recombinant human TF, is characterized by the following features: high physiological stability (the decrease in the activity of the produced enzyme after 50 generations does not exceed 20%) and a high yield of the active product.

Cells grow well on standard nutrient media. The generation time is about 30 min in a liquid LB medium. Round, smooth, yellowish colonies with smooth edges are formed on 2-2.5% Difco Nutrient Agar. When grown on liquid LB and YT media, intense, even turbidity is formed.

Physiological and biochemical signs

The optimum cultivation temperature is from 16 to 42 ° C, the optimum pH is 7.6. Organic compounds (in the form of tryptone, yeast extract) serve as a source of nitrogen.

When these strains are used, their sensitivity to kanamycin (up to 25 μg / ml) is essential. Show resistance to ampicillin (up to 100 μg / ml), due to the presence of plasmids pET21-TF7.

One of the selected clones was designated as Escherichia coli BL21 (DE3) / pET21-TF7, it was grown in 5 ml of liquid LB medium containing 100 mg / L ampicillin to OD _λ600 = 5.0 u.p., an equal volume of sterile 30% glycerol, poured 500 μl into sterile 1.5 ml tubes, frozen and stored at - 70 ° С.

Example 3. Production of BL21 (DE3) / pET21-TF7 biomass and isolation of recombinant human tissue factor

Individual colonies of the recombinant E. coli strain, transformed with the plasmid pET21-TF7, were cultured overnight on an orbital shaker in LB medium at 37 ° C and 180 rpm. The inoculum, in a ratio of 1: 100, was transferred into an Erlenmeyer flask containing LB medium and grown to an optical density (at λ = 600 nm) equal to 0.8. An inducer (isopropyl-β-D-1-thiogalactopyranoside) was added to a final concentration of 0.1 mM. The cells were additionally cultured on a shaker for 12 hours at 25 ° C and 160 rpm. The biomass of bacterial cells was precipitated by centrifugation at 5000 G for 20 minutes. The precipitate was dissolved at the rate of 20 ml per gram in lysis buffer (8% sucrose, 50 mM Tris, 20 mM EDTA, 5% Triton X100 pH = 8.0) using an ultrasonic homogenizer Soniprep 150 Plus (MSE, China ). The pellet containing inclusion bodies was separated by centrifugation (20,000 G for 25 min, 4 ° C) and dissolved in a base buffer (50 mM KH ₂ PO ₄ , 150 mM NaCl, pH = 10.7) containing 8M urea.

After separation, all fractions were analyzed in 12% PAGE for the presence of the target protein. The results showed that all of the target protein is in the insoluble fraction and its percentage of the total amount of proteins is about 10% (Fig. 5).

LIST OF USED SOURCES

1. Grover, S.P. Tissue Factor An Essential Mediator of Hemostasis and Trigger of Thrombosis (Brief Review) / S.P. Grover, N. Mackman // Arterioscler. Thromb. Vasc. Biol.- 2018.- V. 38.- P. 709-725

2. Nemerson, Y. Tissue factor accelerates the activation of coagulation factor VII: the role of a bifunctional coagulation cofactor / Y. Nemerson, D. Repke // Thromb. Res. 1985. V. 40. P. 351-358.

3. Butenas, S. Tissue factor in coagulation: Which? Where? When? /. Butenas, T. Orfeo, K.G. Mann // Arterioscler. Thromb. Vasc. Biol. - 2009. - V. 29. - P. 1989-1996.

4. Williams, J.C. Tissue factor in health and disease / J.C. Williams, N. Mackman // Front Biosci (Elite Ed). 2012. V. 4. P. 358-372.

5. Morrissey, J.H. Tissue factor: an enzyme cofactor and a true receptor / J.H. Morrissey // Thromb. Haemost. 2001 V. 86 P. 66-74.

6. Mackman, N., Tilley R.E., Key N.S. Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis. Arterioscler. Thromb. Vase. Biol. 2007. V. 27. P. 1687-1693.

7. Butenas, S. Tissue factor in thrombosis and hemorrhage / S. Butenas, T. Orfeo, K.E. Brummel-Ziedins, K.G. Mann // Surgery. 2007 V. 142 (4 Suppl.) P. S2-S14.

8. Nemerson, Y. Tissue factor and hemostasis / Y. Nemerson // Blood. 1988. V. 71 (1) P. 1-8.

9. Edgington, T.S. The structural biology of expression and function of tissue factor /T.S. Edgington, N. Mackman, K. Brand, W. Ruf // Thromb. Haemost. 1991 V. 66 (1) P. 67-79.

10. Nakagaki, T. Kisiel Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII / T. Nakagaki, D.C. Foster, K.L. Berkner, W. Kisiel // Biochemistry. 1991. V. 30. 10819-10824.

11. Lawson, J.H. The evaluation of complex-dependent alterations in human factor VIIa / J.H. Lawson, S. Butenas, K.G. Mann // J. Biol. Chem. 1992 V. 267 P. 4834-4843.

12. Komiyama, Y. Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor / Y. Komiyama, A.H. Pedersen, W. Kisiel // Biochemistry. 1990. V. 29. P. 9418-9425.

13. Harlos, K. Crystal structure of the extracellular region of human tissue factor / K. Harlos, D.M. Martin, D.P. O'Brien, E.Y. Jones, D.I. Stuart, I. Polikarpov, A. Miller, E.G. Tuddenham, C.W. Boys // Nature. 1994 V. 370 P. 662-666.

14. McVey, J. H. Review - The role of the tissue factor pathway in haemostasis and beyond / J. H. McVey // Curr. Opin. Hematol. - 2016. - V. 23 (5) P. 453-461.

15. Butenas, S. Comparison of natural and recombinant tissue factor proteins: new insights / S. Butenas // Biological Chemistry. - 2013. - V. 394 (7). - P. 819-829.

16. Boettcher, JM Backbone ¹ H, ¹³ C and ¹⁵ N resonance assignments of the extracellular domain of tissue factor / JM Boettcher, MC Clay, BJ LaHood, JH Morrissey, CM Rienstra // Biomol. NMR Assign. - 2010. - V. 4. - P. 183-185.

17. Ohkubo, Y.Z. Dynamical view of membrane binding and complex formation of human factor VIIa and tissue factor / Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid // J. Thromb. Haemost 2010. V. 8. P. 1044-1053.

18. Fiore, M.M. The biochemical basis for the apparent defect of soluble mutant tissue factor in enhancing the proteolytic activities of factor VIIa / M.M. Fiore, P.F. Neuenschwander, J.H. Morrissey // J. Biol. Chem. 1994. V. 269. P. 143-149.

19. Schaffner, F. Tissue factor proangiogenic signaling in cancer progression / F. Schaffner, N. Yokota, W. Ruf // Thromb. Res. 2012. V. 129 (Suppl. 1). S127-131.

// Prot. Express. Purine- 2002.-V. 26 (3).- P. 386-393.20. Brucato, CL Expression of recombinant rabbit tissue factor in Pichia pastoris, and its application in a prothrombin time reagent./ CL Brucato, CA Birr, P. Bruguera, JA Ruiz,

// Prot. Express. Purine- 2002.-V. 26 (3) .- P. 386-393.

21. WHO Expert Commitee on Biological Standardization, Technical Report series 687, 33 ^d Report, World Health Organization, Geneva, 1983, pp. 81-105.

22. Denson, K.W.E. Comparative studies of rabbit and human recombinant tissue factor reagents / K.W.E. Denson, S.W. Reed, M.E. Haddon, B. Woodhams, C. Brucato, J. Ruiz // Thromb. Res. 1999. V. 94. P. 255-261.

23. Kovacs, M.J. Assessment of the validity of the INR system for patients with liver impairment / M.J. Kovacs, A. Wong, K. MacKinnon, K. Weir, M. Keeney, E. Boyle, M. Cruickshank // Thromb. Haemost. 1994 V. 71 P. 727-730.

24. Kitchen, S. Two recombinant tissue factor reagents compared to conventional thromboplastins for determination of international normalized ratio: a thirty-three-laboratory collaborative study. The Steering Committee of the UK National External Quality Assessment Scheme for Blood Coagulation / S. Kitchen, I. Jennings, T.A. Woods, I.D. Walker, F.E. Preston // Thromb. Haemost. - 1996.- V. 76.-P. 372-376.

25. Smith, S.A. Rapid and efficient incorporation of tissue factor into liposomes. / S.A. Smith, J.H. Morrissey // J. Thromb. Haemost. 2004 V. 2. P. 1155-1162.

26. Paborski, L.R. Purification of recombinant human tissue factor / L.R. Paborski, K.M. Tate, R.J. Harris, D.J. Yansura, L. Band, G. McCray, CM. Gorman, D.P. O_Brien, J.Y. Chang, J.R. Swartz, V.P. Fung, J.N. Thomas, G. Vehar // Biochemistry. 1989. V. 28. P. 8072-8077.

27. Ruf, W. Two sites in the tissue factor extracellular domain mediate the recognition of the ligand factor VIIa / W. Ruf, T.S. Edgington // Proc. Natl. Acad. Sci. USA. 1991. V. 88. P. 8430-8434.

28. Rezaie, A.R. Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calciumdependent antibody / A.R. Rezaie, M.M. Fiore, P.F. Neuenschwander, C.T. Esmon, J.H. Morrissey // Protein Express. Purif. 1992 V. 3. P. 453-460.

29. Stone, M.J. Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity, and physical characterization / M.J. Stone, R. Wolfram, D.J. Miles, T.S. Edgington, P.E. Wright // Biochem. J. 1995. V. 310. P. 605-614.

30. A.J. Austin, C.E. Jones, G. Van Heeke, Production of human tissue factor using the Pichia pastoris expression system / A.J. Austin, C.E. Jones, G. Van Heeke // Protein Express. Purif 1998. V. 13. P. 136-142.

31. Prasad, R. Molecular determinants involved in differential behavior between soluble tissue factor and full-length tissue factor towards factor VIIa / R. Prasad, P. Sen / Phys. Chem. Chem. Phys.- 2017.- V. 19.- P. 22230-22242

32. Studier, F.W. and Moffatt, B.A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-30.

--->

Sequence listing

<110> federal state budgetary educational institution of higher

education "Altai State University"

<120> Recombinant plasmid pET21-TF7, providing synthesis of modified

tissue factor, and Escherichia coli strain BL21 (DE3) pET21-TF7 - producer

recombinant human tissue factor

<160> SEQ ID NO 1

<210> SEQ ID NO: 1

<211> 792

<212> DNA

<213> Artificial Sequence

<220>

<223> Recombinant Tissue Factor TF7 Gene Nucleotide Sequence

person.

<400> 1

ATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGGCACCACCAATACCGTTGCAGCATATAATCTGACCTGG

AAAAGCACCAACTTTAAGACCATTCTGGAATGGGAACCGAAACCGGTTAATCAGGTTTATACCGTTCAGATTAGCACCAAA

AGCGGTGACTGGAAATCCAAATGTTTTTATACCACCGATACCGAATGTGATCTGACCGATGAAATTGTGAAAGATGTGAAA

CAGACCTATCTGGCACGTGTTTTTTAGCTATCCGGCAGGTAATGTTGAAAGCACCGGTAGTGCCGGTGAACCGCTGTATGAA

AATAGTCCGGAATTTACCCCGTATCTGGAAACCAATCTGGGTCAGCCGACCATTCAGAGCTTTGAACAGGTTGGCACCAAA

GTTAATGTTACCGTTGAAGATGAACGTACCCTGGTTCGTCGTAATAACACCTTTCTGAGCCTGCGTGATGTTTTTTGGTAAA

GATCTGATTTACACCCTGTATTATTGGAAGAGTAGCAGTAGCGGTAAGAAAACCGCAAAAACCAATACCAACGAGTTCCTG

ATTGATGTGGATAAAGGTGAGAACTATTGCTTTAGCGTTCAGGCAGTTATTCCGAGCCGTACCGTTAATCGTAAAAGCACC

GATTCACCGGTTGAATGTATGGGTCAAGAAAAAGGTGAATTTCGCGAGATCTTCTATATTATCGGTGCCGTTGTTTTTGTG

GTGATTATTCTGGTTATTATCCTGGCAATTAGCCTGCTGGAACATCATCACCATCACCATTGA (792

nucleotide)

<210> SEQ ID NO: 2

<211> 479

<212> DNA

<213> Artificial Sequence

<220>

<223> Recombinant Tissue Factor Amino Acid Sequence

person.

<400> 2

MASMTGGQQMGRGSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVK

QTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGK

DLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREIFYIIGAVVFV

VIILVIILAISLLEHHHHHH

(263 amino acid residues)

<---

Claims (2)

1. Recombinant plasmid pET21-TF7 of 6171 base pairs (bp), providing the synthesis of recombinant modified human tissue factor, 29.6 kDa in Escherichia coli BL21 (DE3) cells and containing the following structural elements: promoter of bacteriophage T7; a recombinant gene encoding a modified human tissue factor; untranslated transcription termination region of the bacterial operon; phage T7 RNA polymerase terminator; bla sequence (AmpR); ColE1 (ori) sequence; moreover, the recombinant gene of the modified human tissue factor has the nucleotide sequence of SEQ ID NO: 1 from the nucleotide at position 5205 bp. to the nucleotide at position 5996 bp, encoding the target protein with the complete amino acid sequence SEQ ID NO: 2, inserted at the FauND I and CciNI restriction sites into the polylinker region of the pET21a vector containing the promoter and terminator of T7 RNA polymerase. 2. Recombinant Escherichia coli strain BL21 (DE3) / pET21-TF7 - producer of recombinant human tissue factor obtained by transformation with the recombinant plasmid pET21-TF7 according to claim 1.

Images