Fused protein of thioredoxin and infestin 4 domain, method for preparing it, expression plasmid dna coding fused protein, and bacterium escherichia coli transformed by this plasmid dna

FIELD: medicine.

SUBSTANCE: inventions refer to biotechnology and concern a fused protein for the specific inhibition of blood coagulation, an expression plasmid DNA coding this fused protein, a bacterium of the genus Escherichia transformed by this DNA, and to a method for preparing the fused protein. The presented fused protein contains thioredoxin I E.coli and infestine-4 and is characterised by the sequence SEQ ID NO:2. The plasmid DNA contains the sequence SEQ ID NO:1 coding the presented fused protein and controlled by a promoter functioning in a bacterial cell. The method for preparing the above fused protein involves culturing the above bacteria in a nutrient medium, breaking the bacterial cells and purifying the above fused protein with using a metal chelate chromatography and an anion-exchange chromatography.

EFFECT: characterised solutions enables preparing the protein providing the above specificity to be used in blocking the contact activation of blood coagulation by inhibition of the XIIa factor and the absence of inhibition of the Xa factor.

4 cl, 10 dwg, 7 ex

 

The technical field

The invention relates to the field of biotechnology, in particular to the technology of biologically active substances (BAS) by means of genetic engineering.

The prior art.

Biological fluids and tissues of animals and plants contain a large number of protease inhibitors. A large part of the well-studied in the present protein inhibitors contains 60-70 amino acid residues. Along with inhibitors of a wide range of actions, such as alpha-2 macroglobulin, most of the inhibitors reveals certain group specificity. Especially common inhibitors of serine proteases. They proposed to classify, at least 10 families. These include inhibitors of the family of pancreatic trypsinogen inhibitor Marten, family pancreatic secretory inhibitor (Casal).

The family of inhibitors of Casal belongs to the family of inhibitors I1, clan IA classification public MEROPS database [Rawlings, Tolle and Barrett, Evolutionary families of peptidase inhibitors // Biochem J, v.378, p.705-16 (2004)]. Inhibitors of Casal affect serine peptidases family S1, for example, trypsin and elastase. Inhibitors of Casal mainly found in multicellular animals, typical representatives of the family - ovomucoid birds inhibitor acrosin and an elastase inhibitor. Known inhibitors is that family include from one to seven "domains Casal" or "retry Casal" [Williamson, Marion and Wuthrich, Secondary structure in the solution conformation of the proteinase inhibitor IIA from bull seminal plasma by nuclear magnetic resonance // J Mol Biol, v.173, p.341-59 (1984), Laskowski, Kato, Ardelt, Cook, Denton, Empie, Kohr, Park, Parks, Schatzley and et al., Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues // Biochemistry, v.26, p.202-21 (1987)]. The domain structure of Casal includes a significant proportion resveratol polypeptide chains, two short alpha-helix and one beta-layer, consisting of three antiparallel chains [Williamson, Marion and Wuthrich, Secondary structure in the solution conformation of the proteinase inhibitor IIA from bull seminal plasma by nuclear magnetic resonance // J Mol Biol, v.173, p.341-59 (1984)]. In establishing direct intermolecular contacts of the domain of Casal and inhibiting enzyme involved 11 amino acid residues, while 8 of them are hypervariable [Laskowski, Kato, Ardelt, Cook, Denton, Empie, Kohr, Park, Parks, Schatzley and et al., Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues // Biochemistry, v.26, p.202-21 (1987)]. Variation of the amino acids involved in the formation of contacts with inhibiting the enzyme that changes as the inhibition constant, and the specificity of the inhibitor [Empie and Laskowski, Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor interactions with serine proteinases // Biochemistry, v.21, p.2274-84 (1982)].

Known domain of Casal in the protein infestin from the stomach of blood-sucking bug Triatoma infestans specific blocking the activated coagulation factor XII (factor of Hageman, FHA) [Capos, Tanaka-Azevedo and Tanaka, Identification and characterization of a novel factor XIIa inhibitor in the hematophagous insect, Triatoma infestans (Hemiptera: Reduviidae) // FEBS Lett, v.577, p.512-6 (2004)]. Domain blocking FHA, has a sequence number of 4, the remaining domains Casal part intestine specifically inhibit other coagulation factors. Amino acid sequence intestine and boundaries of the domains are listed in the public database UniProt (http://www.uniprot.org/), the record number Q95P16. For an isolated recombinant domain 4 intestine was obtained crystal complex with PHA [Campos, Guimaraes, Medrano, Tanaka and Barbosa, Crystallization, data collection and phasing of infestin 4, a factor XIIa inhibitor // Acta Crystallogr D Biol Crystallogr, v.60, p.2051-3 (2004)] and determined the spatial structure of the complex.

Property infestin-4 to inhibit FHA makes it a promising inhibitor of the contact activation of blood coagulation for use in hamostaseologie, including, as a medicinal product for the treatment and prevention of thrombosis and other diseases associated with hypercoagulation state of the blood coagulation system. Source [Campos, Tanaka-Azevedo and Tanaka, Identification and characterization of a novel factor XIIa inhibitor in the hematophagous insect, Triatoma infestans (Hemiptera: Reduviidae) // FEBS Lett, v.577, R-6 (2004)] the well-known ability infestin-4 to inhibit another factor coagulation system, FHA, with an inhibition constant of 53 nm. This nonspecific effect severely limits the ability of p is imeneniya infestin-4, since FHA is one of the main enzymes of the coagulation cascade. Inhibition of this factor can lead to serious hemostatic complications, such as bleeding [Koscielny J, Beyer-Westendorf J, von Heymann C, Braun J, Klamroth R, Lindhoff-Last E, Tiede A, Spannagl M., Risk of bleeding and haemorrhagic complication with rivaroxaban - Periprocedural management of haemostasis // Hamostaseologie; 32(4):287-93 (2012), Esmon CT, What did we learn from new oral anticoagulant treatment? // Thromb Res. Suppl 1:S41-3 (2012)].

There are two methods of obtaining recombinant infestin-4: in the methylotrophic yeast Pichia pastoris with additional C-terminal peptide in the composition of the fused protein with serum albumin human. Both methods are described in [Hagedorn, Schmidbauer, Pleines, Kleinschnitz, Kronthaler, Stoll, Dickneite and Nieswandt, Factor XIIa inhibitor recombinant human albumin Infestin-4 abolishes occlusive arterial thrombus formation without affecting bleeding // Circulation, v.121, p.1510-7 (2010)]. The productivity of the yeast expression system, infestin-4 is 4 mg/l of culture, productivity systems for the expression of fused protein albumin and infestin-4 in cultured mammalian cells is unknown.

These expression systems infestin-4 in cultured mammalian cells cannot be used due to high cost of cultivation, and well-known expression system infestin-4 in yeast has a too low productivity. These expression systems infestin-4 cannot be used because of respecifies the CSOs inhibition of the produced protein. A method of obtaining the target protein in an active and soluble form with high productivity (patent US 5270181, Peptide and protein fusions to thioredoxin and thioredoxin-like molecules, issued 14.12.1993), which is the expression of a target heterologous protein fused with the protein thioredoxin or thioredoxin protein in host cells. However, the described method of obtaining the target protein does not alter the functional activity of the target protein upon expression in the form of a fused protein with thioredoxin.

Summary of invention

The technical result that can be obtained when implementing the present invention, is effective and specific blocking of the contact activation of coagulation by inhibiting factor Ha and lack of inhibition of factor XA with a simplified technology for protein, providing the specified specificity.

This technical result is achieved by obtaining a fused protein comprising thioredoxin I E. coli and infestin-4, which has a higher specificity of inhibition FHA and having a reduced activity of inhibiting PHA compared with native intestinal-4.

And also by obtaining expression plasmid DNA encoding the aforementioned protein under the control of a promoter that functions in bacteria enoy the cell.

And also by obtaining bacteria-producer mentioned fused protein belonging to the genus Escherichia, transformed the indicated plasmid DNA.

And by creating a method of obtaining mentioned fused protein comprising culturing said bacteria in a nutrient medium, the destruction of bacterial cells and purification of the specified fused protein using metallogenetic chromatography and anion exchange chromatography.

The merits of the proposed solutions

The basis of the proposed solutions are developed by the authors, in particular, plasmid DNA pET32a-Inf4 length 6106 BP encoding a biodegradable protein, including thioredoxin I E.coli, 2 oligoglycines cluster linker plot, sequence specific recognition endoproteinase and located on the C-end of the molecule polypeptide domain 4 intestine T. Infestans. The open reading frame of the specified domain intestine contains optimal for E. coli codons, allowing to increase the level of expression of a heterologous protein due to the efficient broadcast of all amino acids of the polypeptide. The presence of protein-partner maintains the fused protein in a soluble form in the cytoplasm of E. coli, the correct circuit disulfide bonds, allows cleaning fused protein using metalloelastase sorbent, which leads to the considerable increase in the yield of purified fused protein, as well as simplifying the procedures for isolation and purification of the product.

In the process of obtaining the specified protein authors unexpectedly, it was found that infestin-4 in the E.coli bacteria in the form of protein-predecessor or fused protein with thioredoxin significantly increases the specificity of the target protein relative to FHA, lowering the activity of inhibiting the, and can be achieved in high yield expression systems infestin-4.

The term "expression plasmid" means a plasmid DNA containing all of the necessary genetic elements for expression embedded in her gene such as a promoter, terminator. A concrete example of the genetic elements necessary for the expression of fused protein thioredoxin and domain 4 intestine comprising the expression cassette according to the present invention is, but not limited to, the promoter is an RNA polymerase of bacteriophage T7.

The DNA fragment coding for the recombinant domain 4 intestine T.infestans according to the present invention is, for example, a synthetic gene that encodes a recombinant domain 4 intestine T.infestans in the composition of the fused protein comprising a sequence encoding ottsepleny N-terminal protein partner, which, in turn, thioredoxin I E.coli, oligoglycines clusters, sequence recognition specification is eskay endoproteinase and domain 4 intestine T.infestans. The specified DNA fragment can be obtained by PCR (see Example 1, Fig 1). Also mentioned DNA fragment can be obtained using cloning technology company Sloning BioTechnology, described in PCT application WO 2005071077.

To ensure effective translation of the cloned gene in E. coli, preferably in the sequence that encodes a protein, and in particular recombinant domain 4 intestine .infestans, all rare codons were replaced by synonyms common genes in E. coli codons. The sequence of Trx-Inf4 encoding domain 4 intestine .infestans in the composition of the fused protein according to the present invention, presented in the sequence Listing under the number SEQ ID NO: 1.

The amino acid sequence of the fused protein containing the domain 4 intestine .infestans according to the present invention, presented in the sequence Listing under the number SEQ ID NO: 2.

Amino acid sequence of the intact recombinant domain 4 intestine .infestans is a sequence number SEQ ID NO: 2 182 without first amino acids (i.e. amino acids 183 - 238).

DNA fragments that encode essentially the same protein can be obtained, for example, by modifying the nucleotide sequence of the DNA fragment (SEQ ID NO: 1)encoding a protein containing the domain 4 infestin the .infestans, for example, using the method of site-directed mutagenesis, so that one or more amino acid residues at a specific site will be deleterows, substituted, inserted or added. The DNA fragments, modified as described above, can be obtained using traditional processing methods with the aim of obtaining mutations. DNA fragments that encode essentially the same protein can be obtained by expression of the DNA fragments having the mutation described above, in the appropriate box.

Indicators of functional activity, in which it is considered that the resulting protein has properties of an inhibitor of factor GA is determined by its ability to inhibit blood clotting initiated by "internal" path. For example, the activity domain 4 intestine Triatoma infestans can be detected in coagulopathies test "kaolin time", as described in Example 6. It is believed that the variant protein has properties of domain 4 intestine .infestans provided that this option increases the clotting time of blood plasma is not less than 30% at a concentration not higher than 5 μm.

The term "specificity of inhibition FHA" means the properties of the inhibitor PHA, which consists in inhibition FHA with high efficiency (the inhibition constant of the order of 1 nm or below) and the absence of noticeable inhibi the Finance (inhibition constant is much greater than 1 nm, preferably greater than 1 μm) of other proteases of the coagulation, such as factors XIa, IXa, XA, IIa (thrombin), and VIIa. Native infestin-4 inhibits PHA with a rate constant of 0.1 nm, with protein inhibits the with the constant of 53 nm. Protein of thioredoxin and infestin-4 has the same inhibition constant FHA as native inhibitor, but the inhibition constant of the is 2.5 μm, i.e. up to 50 times higher. Thus, the resulting protein has a high specificity to FHA compared to the native protein.

The expression plasmid according to the present invention contains a DNA fragment encoding a protein containing domain 4 intestine .Infestans and including a sequence encoding ottsepleny N-terminal protein partner, including thioredoxin I E.coli, 2 oligoglycines cluster (6 and 10 histidine), 2 sequence specific recognition endoproteinase (DDDDK) and located at the end of the fused protein directly after the last site recognition DDDDK domain 4 intestine .infestans, under the control of a promoter functional in a bacterial cell. As recombinant plasmids according to the present invention may be used various plasmids, capable of expression in a cell of the recipient, such as plasmids pBR322, pMW119, pUC19, RETA, pET28b and the like, but the list of plasmids much more than knowledge of them.

One of the options for implementing the present invention is a plasmid pET32a-Inf4 size 6106 BP, which consists of:

1) fragment HindIII-NheI (1-204) with a length of 204 BP, containing the section that encodes domain 4 intestine .infestans and merged with it in a frame sequence of the FLAG-epitope, which includes the site of recognition of enterokinase

2) fragment NheI-NcoI (205-245)-length 41 BP, containing the section that encodes the peptide SAGHHHHHHHHHHG containing decamethylenebis cluster

3) slice NcoI-HindIII (246-6106) vector rate(+) length 5861 BP, containing the region of the beginning of replication of plasmids pBR322, gene RNA-organizing protein Rop, the site of initiation of replication of bacteriophage f1, sequence, encoding beta-lactamase, the promoter is an RNA polymerase of bacteriophage T7; plot termination of transcription; the sequence encoding the repressor of Lac operon; the sequence encoding thioredoxin I E.coli, exegetically tag and the site of cleavage by enterokinase. The indicated plasmid contain a unique recognition sites of the restriction endonucleases: HindIII (1), NheI (205), NcoI (246), XbaI (736), PciI (3656), PvuI (4919).

The structure of the plasmid pET32a-Inf4 is shown in Figure 3 and in SEQ ID 3.

Using a plasmid can be transformed bacterial cell, preferably a bacterium of the genus Escherichia, receptive to such transformation specified by the plasmid. Selecting specific to EDI is not critical, because the methodology and methods of transformation are well known to the person skilled in the art. Although depending on the type of cells and culture conditions the received transformant the level of expression of a fused protein containing domain 4 intestine .infestans may vary, the fact that expression of the target protein will be subject to the successful transformation of the cells of the recipient.

"Transforming cells with plasmid" means the introduction of plasmids into the cell using methods well known to the person skilled in the art. Transformation of this plasmid is required for expression of the gene encoding the protein according to the present invention, and protein synthesis in the bacterial cell. Methods of transformation include any of the standard methods known to a person skilled in the technical field, for example the method described in Jac A. Nickoloff, Electroporation Protocols for Microorganisms (Methods in Molecular Biology) //Humana Press; 1st edition (August 15, 1995).

According to the present invention, the bacterial cell producing fused protein thioredoxin and domain 4 intestine .infestans" means a bacterial cell that is capable of production and accumulation of fused protein thioredoxin and domain 4 intestine .infestans, when the bacterial cell according to the present invention are grown in a specified medium. Used herein, the term "tank is Arianna cell - producer fused protein thioredoxin and domain 4 intestine .infestans" also means a cell which is capable of accumulating protein of thioredoxin and domain 4 intestine .infestans in the amount of not less than 2 mg/l of culture, more preferably not less than 20 mg/l culture. The specified protein of thioredoxin and domain 4 intestine .infestans accumulates in the specified cell is preferably in the cytoplasm in a soluble form.

Preferably using bacteria belonging to the genus Escherichia, for transformation with recombinant plasmid containing the DNA fragment, encoding a protein of thioredoxin and domain 4 intestine .infestans.

The term "bacterium belonging to the genus Escherichia can mean that the bacterium belongs to the genus Escherichia according to the classification known to a specialist in the field of Microbiology. As examples of the microorganism belonging to the genus Escherichia can be mentioned Escherichia coli (E. coli).

The range of bacteria belonging to the genus Escherichia, is not limited in any way, however, for example, bacteria, described in the book Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, table 1), can be given as examples.

A concrete example of the strain of the recipient to obtain the product of fused protein thioredoxin and domain 4 intestine .infestans according infusion is to him the invention is, but are not limited to, Escherichia coli strain BL21[DE3].

The Escherichia coli strain BL21[DE3] is characterized by the following cultural-morphological, physiological and biochemical characteristics and genetic traits.

Cultural and morphological characteristics of strain: gram-negative rods that form filaments; on agar medium - large whitish colonies with rough edges. The activity of the strain is determined using densitometry of electrophoregram. The strain is stored in the following conditions: the environment Lurie-Bertrand, 1% glucose, 10% glycerol. Strain propagated in the following conditions - environment Lurie-Bertrand, 1% glucose, ampicillin 50 mg/ml

Genetic strain. The genotype of strain - F-ompT gal dcm lon hsdSB(rB-mB+) λ(DE3[lacI lacUV5-T7 gene 1 ind1 sam7 nin5]).

Transformation of Escherichia coli strain BL21[DE3] the plasmid pET32a-Inf4 results of the producer strain BL21[DE3]/pET32a-Inf4, which provides a synthesis of the recombinant fused protein thioredoxin and domain 4 intestine (Trx-Inf4) .infestans more than 5% of the total protein content of the cells.

The Escherichia coli strain BL21[DE3]/ pET32a-Inf4 encodes a protein Trx-Inf4 consisting of the amino acid sequence of domain 4 intestine .infestans and merged with it in the box protein partner (thioredoxin E. coli)containing the site of cleavage by enterokinase directly re the first amino acid domain 4 intestine .infestans.

A method of obtaining a fused protein Trx-Inf4 consisting of the amino acid sequence of domain 4 intestine .infestans and merged with it in the box protein partner of thioredoxin I E.coli according to the present invention includes culturing the above-described bacteria in a nutrient medium suitable for the cultivation of these prokaryotic cells, the induction of gene promoter fused protein Trx-Inf4, biomass harvesting cells, destruction, separation of insoluble proteins and debris, cleaning the slit protein Trx-Inf4 metallogenetic chromatography in native conditions and subsequent anion-exchange chromatography.

Features of plasmids and the results of practical application is shown in the following figures.

Brief description of figures:

1 shows a diagram of the Assembly of the synthetic gene domain 4 intestine Triatoma infestans of oligonucleotide primers and the scheme of obtaining expression plasmids p10E - Inf4 and pET32a-Inf4. The following symbols are used:

Inf4 - domain 4 intestine Triatoma infestans, the corresponding domain 4 type Casal. In parentheses are the first and last amino acids of fragments. The dotted line indicates polymerase chain reaction, solid - restriction and ligation of DNA fragments.

Figure 2 shows a map of the expression plasmid p10E-Inf4. The following symbols are used: "pBR322" - the replication origin of the plasmid is pBR322; "ROP" - gene RNA-organizing protein Rop, controlling chopinot plasmids; "fl ori" - the site of initiation of replication of bacteriophage f1; "KanR2" - gene resistance to kanamycin; "T7 prom" - promoter RNA polymerase of bacteriophage T7; "T7 term - the area of termination of transcription of the target gene; "lacI" - gene repressor of Lac operon; Inf4 - open reading frame (ORF) of the polypeptide domain 4 intestine Triatoma infestans; "FLAG - epitope monoclonal antibodies FLAG; "EK-PRO - site recognition of enterokinase, "10TH tag" - detachable N-terminal peptide, including decamethylenebis cluster, the FLAG epitope and the site of recognition of enterokinase; stop - block stop-codons. Arrows indicate the directions of transcription of the genes in parentheses are the numbers of the first and the last nucleotide fragments. Identifies the recognition sites of restriction endonucleases in parentheses are the numbers of nucleotides at the point of cutting.

Figure 3 shows a map of the expression plasmid pET32a-Inf4. The following symbols are used: "pBR322 origin - region start replication of plasmids pBR322; f1 origin" - the site of initiation of replication of bacteriophage f1; "BIa (AmpR)" sequence that provides resistance to bacteria to ampicillin; "T7 promoter" is a promoter of RNA polymerase of bacteriophage T7; "T7 terminator - the area of termination of transcription; "lacI" - the sequence encoding the repressor of Lac operon; "Trx-Inf4" - OR the fused protein; "Inf4" - LFS domain 4 intestine Triatoma infestans; "Trx" - OPC-thioredoxin I E.coli; "10xHis" - decamethylenebis cluster; "6xHis" - exegetically cluster; "EC-PRO - part of the encoding site recognition by enterokinase; "FLAG - epitope monoclonal antibodies FLAG; stop - block stop-codons. Arrows indicate the directions of transcription of the genes in parentheses are the numbers of the first and the last nucleotide fragments. Identifies the recognition sites of restriction endonucleases in parentheses are the numbers of nucleotides at the point of cutting.

Figure 4 shows electrophoregrams total protein from cells of the producer strain BL21[DE3]/pET32a-Inf4 (4 colonies) and a control strain BL21 [DE3]/pET32a induction IPTG.

Induction of 1 mm IPTG, 30°C; 0.4 and 16 o'clock Legend: Inf cl. - the corresponding colony producer strain BL21[DE3]/pET32a-Inf4, Trx ( -) - colony control strain BL21[DE3]/pET32a, 0h - 16h - time induction h; M - molecular mass marker. The molecular weight marker bands are indicated in kDa. The position of the target protein and the control protein partner indicated by arrows. Reducing conditions, the color of the colloidal Kumasi blue. The calculated productivity for strain BL21[DE3]/pET32a-Inf4: 20-40 mg/l, the proportion of the target protein: >5% (densitometry of the gel).

Figure 5 shows electrophoregrams protein fractions obtained during the isolation and one-step chromatographic purification of fused protein Trx-nf4 and control protein partner Trx.

Legend: M - marker molecular mass, molecular mass bands are indicated on Figure 4, "IP" - total protein after induction, "IB" - insoluble proteins, "SO" soluble proteins, "7R" - precipitate after thermocoagulation, "7S" - supernatant after thermocoagulation, "AS" supernatant during the precipitation of ammonium sulfate, "IM" - reconstituted precipitate after ammonium sulfate precipitation, FF is the fraction of leakage when metallogenetic chromatography, "100", "200", "500" - faction eluates with metallogenetic column corresponding concentration of imidazole, E - fraction elution EDTA-Na, NA - fraction elution regeneration column NaOH. Non conditions, the color of the colloidal Kumasi blue. The position of the target protein Trx-Inf4 arrow.

Figure 6 shows electrophoregrams protein fractions obtained during the gradient elution fused protein Trx-Inf4 with sorbent Capto q Notation: "M" is a marker of molecular weights, molecular weight bands are indicated on Figure 4, "200" - paleoceanic Trx-Inf4 before applying; "FF" is the fraction of leakage when metallogenetic chromatography; F1"-"F8" - corresponding to the collected fractions of the eluate. Non conditions, the color of the colloidal Kumasi blue. The position of the target protein is indicated by an arrow.

7 shows the chromatogram OF HPLC analysis of the purified fused protein Trx-Inf4 and the results of integration of the peaks. To the cash detection 280 nm, the purity of the protein 99,06%.

On Fig shows electrophoregrams track with purified Trx-Inf4 and the results of densitometry. Reducing conditions, the color of the colloidal Kumasi blue, the purity of the protein 99,48%.

Figure 9 shows a plot of clotting time of blood plasma in kaolin test concentration in plasma Trx-Inf4.

Figure 10 shows a plot of the activity of the determined by the rate of cleavage of a chromogenic substrate, the concentration of Trx-Inf4.

The present invention will be described in more detail below with reference to the following, not limiting the present invention, examples.

Example 1. Plasmid DNA isolation pAL-Inf4

For amino acid sequences, including 56 amino acids of domain 4 intestine Triatoma infestans (amino acids 167-222 of recording Q95P16 public databases UniprotKB located at the address http://www.uniprot.org/uniprot/Q95P16 with the added amino acid N-terminal peptide (SEQ ID NO: 4, ASDYKDDDDK), containing the sequence recognition by enterokinase was held back translation into a sequence of DNA nucleotides. If this were used codons optimal for the expression of this gene in E. coli class, and was also optimized the structure of the gene on secondary structure of mRNA, GC composition, provided no unwanted regulatory elements (for example, lack the of nutrena binding sites of the ribosome), and the absence of long repeats, palindromes. In the DNA sequence were also included stop codons and the recognition sites of restriction endonucleases. The obtained nucleotide sequence shown in SEQ ID NO:5. Physical fragment EKsite-Kaz-4 (SEQ ID NO:5) was collected by polymerase chain reaction (PCR) of the overlapping oligonucleotide primers AS-KAZ-1F AS-KAZ-2R AS-KAZ-3F AS-KAZ-4R AS-KAZ-5R shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, respectively.

PCR was performed on the device Terzic MC2 (DNA-technology, Russia). Preparative reactions were performed in a volume of 50 μl. Preparing the incubation mixture of the following composition: 1x buffer for thermostable DNA polymerase; 10 pmol of each primerno of the oligonucleotide; 2 mm each deoxyribonucleotide; 2 units of thermostable DNA polymerase. On top of this mixture was layered with 50 μl of mineral oil and amplification were according to the scheme: 1 cycle of denaturation 94°C, 3 min; 25 cycles of denaturation 94°C, 30 sec, annealing 62°C, 30 sec, extension 72°C, 120 sec; 1 cycle - extension 72°C, 10 min PCR Products were isolated from 1% agarose gel set "Wizard SV Gel and PCR Clean-Up System (Promega, USA) according to the manufacturer's Protocol, and then provided in the T-vector pAL-TA (Evrogen, Russia) using DNA ligase of phage T4 and standard buffer solution ("Fermentas", Lithuania). Ligation led in the volume of 10 μl at a molar ratio of age of the ora and insert 1:10, for 2-20 hours at room temperature. Received ligase mixture was used to transform cells of E. coli strain DH5, with genotype F - φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk, mk+) phoA supE44 λ - thi-1 gyrA96 relA1. To do this, 200 µl of frozen E. coli cell suspension was added 5 μl of ligase mixture, incubated on ice for 20 minutes for adsorption of plasmid DNA was heated to 42°C for 45 seconds and incubated on ice for 5 minutes. Then add 800 ál of nutrient broth LB and incubated at 37°C for 60 minutes. After incubation the suspension was transferred to a Petri dish containing solid agar medium containing ampicillin at a concentration of 100 μg/1 ml of the agar and placed in a thermostat at 37°C for 18 hours. Colonies of E. coli, selected by blue-white screening, were analyzed by PCR from clones using primers to sequences recipient plasmids: M13dir (SEQ ID NO:11) and M13rev (SEQ ID NO:12). The selected clones was increased in 5 ml of nutrient broth LB and perform the selection plasmid DNA assay kit "Wizard Plus SV Minipreps" (Promega, USA) according to the Protocol of the manufacturer, the primary nucleotide sequence of the constructs was verified by PCR-sequencing using primers T7prom (SEQ ID NO:13) and SP6 (SEQ ID NO:14).

Example 2. Obtain expression plasmid DNA p10E-Inf4.

The recipient plasmid p10E was restrictively consistently each end of the nucleases NheI and HindIII. First, aliquots of the plasmid were incubated 2 hours at 37°C with each of restricts, was controlled by agarose gel electrophoresis full linearization of the plasmid, then added a second restriction enzyme and incubated for another 2 hours. Then the samples were combined, enzymes iactiveaware by heating at 65°C for 20 minutes and was conducted by dephosphorylation by alkaline phosphatase ("Fermentas", Lithuania) according to the manufacturer's Protocol. Alkaline phosphatase iactiveaware heating to 85°C for 20 minutes. Restricion and dephosphorylated plasmid was perioadele 3 volumes of ethanol, centrifuged for 10 minutes at the speed 13200 rpm at room temperature, washed the precipitate with 70% ethanol, dissolved in water and used for the production of the ligation reaction in a working concentration of 10-2µg/µl. The reaction ligating the purified fragment NheIHindIII plasmid pAL-Inf4 corresponding to minigene domain 4 intestine and recipient plasmid was performed as described in example 1. Then spent the transformation of cells of E. coli strain DH5 alpha, as described above. Colonies of E. coli were analyzed by PCR from clones with primers T7prom (SEQ ID NO:13) and T7term (SEQ ID NO:15).

The selected clones was increased in 5 ml of 2xYT medium with kanamycin, spent isolation of plasmid DNA with a set of "Wizard Plus SV Minipreps" (Promega, USA). Using PCR-sequencing for construction p10E-Inf4 defined well leonidou sequence of the two complementary DNA strands for the insert using primers T7prom and T7term. In the sequencing established that the drug plasmids do not contain mutations in the paste area, that is encoded with the correct gene sequence domain 4 intestine. Map expression constructs shown in Figure 2 and in SEQ ID NO:16, amino acid sequence of the expression product is shown in SEQ ID NO:17.

Example 3. Obtain expression plasmid DNA pET32a-Inf4.

The recipient plasmid rate (+) (Novagen, USA) was restrictively consistently each endonucleases NcoI and HindIII as described in example 2. Donor plasmid p10E-Inf4 was restrictively NcoI and HindIII and purified from a 1.5% agarose gel set "Wizard SV Gel and PCR Clean-Up System (Promega, USA). The selected fragments are ligated, transformed legacy in E. coli strain DH5-alpha and produced a selection of clones as described in example 2. The selected clones was increased in 5 ml of 2xYT medium-Amp, was isolated plasmid DNA kit "GeneJET Plasmid Miniprep Kit" and determined the nucleotide sequence of DNA by PCR-sequencing with primer T7term. Card design shown in Figure 3, the sequence in SEQ ID NO:3, amino acid sequence of the target protein is shown in SEQ ID NO:2.

Example 4. Obtaining strains producing E. coli BL21[DE3]/p10E-Inf4 and E. coli BL21[DE3]/pET32a-Inf4, assessing the productivity of strains producing and localization of the target protein.

To obtain strains-producers of the slit is about protein thioredoxin E. coli linker segment and domain 4 intestine (Trx-Inf4) and protein-precursor 10TH-Inf4 design obtained in examples 2 and 3 was used to transform competent cells of Escherichia coli BL21(DE3) (genotype F - otrt hsdSB(r-m-) gal dcm (DE3)).

To obtain the strain E. coli BL21[DE3]/p10E-Inf4-producer protein precursor domain 4 intestine Triatoma infestans in the composition of cells of E. coli strain BL21[DE3] transformed the expression plasmid p10E-Inf4.

To obtain the strain E. coli BL21[DE3]/ pET32a-Inf4-producer fused protein Trx-Inf4 cells of E. coli strain BL21[DE3] transformed the expression plasmid pET32a-Inf4.

Similarly, the received control strain E. coli BL21[DE3]/pET32a-producer of thioredoxin I E. coli (Trx), transforming cells of E. coli BL21[DE3] the plasmid rate.

The E. coli transformants were sown on agar 2xYT medium with addition of glucose to 2% and selective antibiotics - 30 µg/ml kanamycin for BL21[DE3]/p10E-Inf4 or 50 μg/ml ampicillin for BL21[DE3]/pET32a-Inf4 and BL21[DE3]/pET32a; conducted the induction of expression of a target protein for four randomly selected colonies with typical phenotype. Colonies were pokasivali in nutrient broth with the addition of the appropriate antibiotic and glucose to 2% for 14 hours, inoculable a new portion of the nutrient medium at a ratio of 1:100, raise culture to achieve an optical density of 1-1,5 PU, induced isopropylthio-R-D-galactoside and who has antivirali another 16 h, taking aliquots of the suspension after 4 h induction. After the cultivation, the precipitated cells were separated by centrifugation, resuspendable cells in a solution of 10 mm Tris-HCl, 2 mm EDTA-Na, 0.1% Triton-X100, 10 μg/ml of lysozyme in the ratio of 10 ml per 1 g of cell paste was kept in suspension for 30 min on ice and carried out the destruction of the cells by ultrasonic disperser to the disappearance of the apparent viscosity of the suspension. Taking samples for electrophoretic analysis, share them soluble and insoluble protein fraction by centrifugation in microcentrifuge additionally resuspendable precipitate in the same solution and precipitated by centrifugation. The results of electrophoretic analysis of total protein for strain BL21[DE3]/pET32a-Inf4 is shown in Figure 4. Electrophoretic mobility of the target protein corresponds to the calculated values. According to gel electrophoresis of protein fractions (Figure 5) target protein Trx-Inf4 and control protein-partner Trx were almost completely localized in the soluble fraction of proteins. In the case of strain BL21[DE3]/p10E-Inf4 electrophoretic analysis showed no visible product of the expression.

Example 5. Isolation and purification of fused protein Trx-Inf4

Producing strains BL21[DE3]/pET32a-Inf4 and the control strain BL21[DE3]/pET32a were sown from the Museum loop debilitating stroke on a Petri dish with agar LB medium, with the holding of 30 μg/ml kanamycin and 1% glucose, raised 14 hours at 37°C. a single colony of each strain was transferred into 5 ml of liquid medium 2xYT containing 100 μg/ml ampicillin and 1% glucose and grown on a shaker for 14 hours at 37°C. the Contents were inoculable 2 flasks containing 250 ml of medium Terrific broth, optionally containing 100 μg/ml ampicillin and 0.1% glucose, raised on the rocking 3.5 hours at +37°C, was selected samples of bacterial suspensions for analysis, was added IPTG to a final concentration of 1 mm and grown for another 4 h at +30°C. the precipitated cells were separated by centrifugation, resuspendable in 20 ml of solution A (50 mm Tris pH of 7.4, 2 mm EDTA)was added to the lysozyme, 0.1 mg/ml and Triton X100 0.1%, incubated for 30 min on ice. Spent the destruction of cells and genomic DNA by using an ultrasonic disperser heartbeats for 10 seconds before disappearing to the high viscosity of the suspension. Separating the precipitate by centrifugation 10 min at 20000 rpm were thermocoagulation impurity proteins in the resulting clarified the lysate incubation at a temperature of +70°C (the maximum possible for Trx-Inf4) for 10 minutes precipitate was Separated by centrifugation for 10 min at 5000 rpm Besieged target proteins by addition of an equal volume of a saturated solution of ammonium sulfate, kept for 30 min on ice, centrifuged 20 min at 5000 rpm, the supernatant was discarded. Sediment suspended in 10 ml of solution B (50 mm Tris-HCl, 500 mm NaCl, 10 m is imidazole, pH 7.4), denaturated proteins were separated by centrifugation for 20 min at 5000 rpm, the Supernatant was filtered through a disc filter with pores of 0.45 μm and was applied to a 1 ml column with sorbent Chelating Sepharose Fast Flow (GE Healthcare, USA), containing chelated Nickel ions, and balanced solution Century. Drawing conducted at a speed of 0.5 ml/min Breakthrough was collected for further analysis. Conducted step elution of adsorbed protein solution with a concentration of imidazole 100, 200, 500 mm at a flow rate of 1 ml/min was Removed immobilized Nickel ions solution B+50 mm EDTA-Na, pH 8.0. Regenerates the column with 10 ml solution of 0.1 M sodium hydroxide. The eluate when washing with sodium hydroxide immediately neutralized with 1.2 mole equivalent of acetic acid. Aliquots of all protein fractions obtained were analyzed by using LTO-page in non conditions (Figure 5).

Final purification of the target protein was performed using anion exchange chromatography using dextranomer sorbent Capto Q (GE Healthcare, USA). A column volume of 1 ml balanced solution A (50 mm Tris-HCl, 50 mm NaCl, pH 7.4), washed with 5 volumes of solution B (50 mm Tris-HCl, 500 mm NaCl, pH=7,4) and again washed with 5 volumes of solution A. Solution procedendo Trx-Inf4 after metallogenetic chromatography was diluted with water, treated 10 times, and put on urovnoveshennuyu Capto Q at a speed of 3 ml/min The column was washed with 10 volumes of solution A and perform a gradient elution from 0 to 100% solution B over 20 min at a flow rate of 1 ml/min was Collected fractions containing the protein according to the UV-detector, and analyzed them using LTO-page (6).

Fractions containing the target protein were pooled and concentrated by ultrafiltration using a disposable cell Vivaspin 6 PES 5 kDa (Sartorius Stedim, Germany) to a final volume of 1.5 ml of the Concentrated solution was transferred into microprobing added preservative thimerosal (Sigma, USA) to a final concentration of 0.02%, was divided into aliquots of 200 μl and transferred to storage at minus 20°C.

Example 6. Analysis of the purified fused protein Trx-Inf4

The resulting solution of purified Trx-Inf4 measured concentration of the basic substance and the proportion of polypeptide impurities using obremenitve chromatography. Used column SOURCE RPC 4.6 ST (GE Healthcare, USA) mobile phase A - 5% acetonitrile, 0.1% of triperoxonane acid in water; B : 80% acetonitrile, 0.1% of triperoxonane acid in water, gradient program - 5 min 100% solution A, from 0 to 100% solution B over 40 min, 3 min 100% solution B from 100 to 0% solution B for 1 min, 3 min 100% solution A, the rate of 1 ml/min, column temperature 40°C. The volume of injected sample - 15 ál of the sample-preparation - addition of acetonitrile to 10% and tuttorosso acid to 1%, dilution of the sample 1.11 times. Detecti is conducted at wavelengths of 280 and 214 nm.

The purity of the basic substance was similar when the analysis data of both channels detection and amounted to more than 98%. The chromatogram for channel 280 nm is shown in Fig.7.

When processing biomass obtained from 500 ml of bacterial suspension was obtained 24 mg of purified protein Trx-Inf4 (according to RP-HPLC, assuming 80% elution of the basic substance in the analysis), which corresponds to the productivity of the developed system 48 mg fused protein from one liter of bacterial culture grown in flasks.

To identify related impurities, which could co-elute with the peak of the main substances by RP-HPLC, conducted electrophoretic analysis of purified Trx-Inf4 in reducing conditions, followed by densitometry of electrophoregram. Was detected one band admixture with volume densitometrical peak around 0.5% of the volume densitometrical peak of the basic substance (Fig).

Functional activity of Trx-Inf4, i.e. the ability to slow the clotting of blood plasma on "internal" path due to the inhibition of factor Ha, investigated using kaolin test". Used a suspension of kaolin and liofilizovannye normal plasma production NGOs to Rena (Russia); the measurements were carried out on semi-automatic coagulometer ThromboScreen 400 (Pacific Hemostasis, USA) according to the instructions of the manufacturers of the device and is of eagent. The sample Trx-Inf4 was introduced into the plasma for 1 min before addition of a suspension of kaolin in the amount of not more than 10 μl. When making Trx-Inf4 in the plasma at a final concentration of 0.4 mg/ml (7.5 μm) clotting time was 192 to control Trx protein at a final concentration of 0.4 mg/ml - 65, for the intact plasma - 57 C. Thus, it was found that protein Trx-Inf4 in high concentrations can slow the clotting time of plasma in the kaolin test 3 times. To establish actionable when coagulopathies diagnostics working concentration of Trx-Inf4 made measurements kaolin time for different concentrations made Trx-Inf4 (Fig.9). The clotting time of the plasma increases rapidly with increasing concentration of Trx-Inf4 from 150 to 300 nm and with a further increase in the concentration of the inhibitor changes slightly, which corresponds to full lock activated on the surface of particles of kaolin factor XII and subsequent non-specific inhibition of other trypsin-like coagulation factors.

Example 7. The definition of anti-FHA activity of the protein Trx-Inf4.

To determine the inhibitory activity of Trx-Inf4 against factor XA (FHA) used the cleavage reaction of the (Hematologic Technologies Inc., USA) - specific chromogenic substrate S-2765 (Chromogenix, Sweden). In cells of the tablet has consistently placed 50 μl of RA the creators of the (final concentration 0.5 nm) in buffer (50 mm Tris-HCl, 130 mm NaCl and 0.5% BSA, pH 8.3), 50 μl of a solution Trx-Inf4 in the buffer C or solvent (0.3 M Hepes, pH 7.4). Before beginning the reaction mixture was incubated for 15 minutes at 37°C, after which was added 100 μl of a solution of a chromogenic substrate (final concentration of S-2765 500 μm). The initial rate of hydrolysis of the substrate was measured spectrophotometrically at a wavelength of 405 nm. Determined the percentage of inhibition of the rate of hydrolysis in the presence of various concentrations of Trx-Inf4 (relative to the speed of the sample without inhibitor) and the concentration value of Trx-Inf4, reduce the rate of hydrolysis by 50% (IC50). IC50 was 7.5 μm. The inhibition constant KI with respect to the can be calculated by the equation of Cheng-Prusoff (KI = IC50/(1+S/Km)), where S is the substrate concentration, Km is the constant of Michaelis-Menten substrate relative to FHA, component 250 μm. KI for Trx-Inf4 in relation to PHA was 2.5 μm, i.e. about 50 times higher than for native infestin-4, and about 10 times higher than the working concentration, practically applicable when coagulopathies diagnosis. Thus, protein Trx-Inf4, compared with native intestinal-4 is a more specific inhibitor PHA.

The results showed that the inclusion in the sequence of the resulting polypeptide N-terminal protein-partner thioredoxin in combination with a synthetic genome domain 4 infest is on, coded optimal for E. coli codons, retains the functional properties of domain 4 intestine, which consists in inhibition FHA and contact activation of coagulation, and significantly increases the level of expression, ensures the correct folding of heterologous protein domain in bacterial cytoplasm.

The advantage of this invention is to increase the specificity of the resulting inhibitor of Trx-Inf4 to FHA compared with native intestinal-4, which is that Trx-Inf4 inhibits the 50 times weaker. This suggests that the use of a fused protein thioredoxin and infestin-4 in the range of concentrations at which it is effectively inhibited by contact activation will not occur undesirable effect of non-specific inhibition of other proteases of the coagulation, in addition to FHA.

The advantages offered by the strain E. coli BL21[DE3] consist in the use of bacteria phenotype Lon Otrt, which excludes the possibility of proteolytic cleavage of the synthesized de novo recombinant polypeptide and contamination secreted protein most active proteases in E. coli. Integrated into the genome of the strain of the recipient gene RNA polymerase of bacteriophage T7 under the control of the lacUV5 promoter using T7-lac promoter and T7 terminator in the plasmid leads to a rapid and effective products is AI protein.

While this invention is described in detail with reference to Examples, for a specialist in a specified field of technology it is obvious that can be done various changes and produced equivalent replacement, and such modifications and substitutions are within the scope of the present invention.

1. Protein for specific inhibition of blood coagulation, including thioredoxin I E. coli and infestin-4, characterized by the sequence SEQ ID NO: 2, while it has a high specificity of inhibition FHA and has a lower inhibitory activity to the compared with native intestinal-4.

2. Expression plasmid DNA,
contains the sequence of SEQ ID NO: 1, coding for a protein according to claim 1 and under the control of a promoter functional in a bacterial cell, preferably the promoter is an RNA polymerase of bacteriophage T7, and it contains one or more of these areas: the area started replicating plasmids, gene RNA-organizing protein Rop, the site of initiation of replication of bacteriophage f1, sequence, encoding beta-lactamase, the site of termination of transcription, a sequence encoding the repressor of Lac operon, the sequence encoding thioredoxin I E. coli sequence, encoding polyhistidine tag.

3. The bacterium belonging to the genus Eschrichia, the transformed plasmid DNA according to claim 2, for producing fused protein according to claim 1.

4. A method of obtaining a fused protein according to claim 1, comprising cultivating the bacterium according to claim 3 in a nutrient medium, the destruction of bacterial cells and purification of the fused protein using metallogenetic chromatography and anion exchange chromatography.



 

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16 cl, 5 dwg

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