RU2495933C2 - PLASMID FOR EXPRESSION IN BACTERIA CELLS OF GENUS Escherichia OF PRECURSOR OF RECOMBINANT FRAGMENT OF HUMAN TISSUE PLASMINOGEN ACTIVATOR (TPA) (RETEPLASE), BACTERIA BELONGING TO GENUS Escherichia, - PRODUCER OF PRECURSOR OF RECOMBINANT FRAGMENT OF HUMAN TPA (RETEPLASE) OR [-1] METHIONYL - FRAGMENT OF HUMAN TPA (RETEPLASE), PRECURSOR OF RECOMBINANT FRAGMENT OF HUMAN TPA (RETEPLASE), METHOD OF PRODUCTION OF PRECURSOR OF RECOMBINANT FRAGMENT OF HUMAN TPA (RETEPLASE) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a method of production of precursor of a recombinant fragment of human tissue plasminogen activator (tPA) (reteplase) using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a precursor of the recombinant fragment of human tissue plasminogen activator (tPA) (reteplase), comprising a sequence encoding a cleavable N-terminal peptide comprising decahistidine cluster and sequence of enterokinase recognition fused in frame, or a fragment of DNA encoding the [-1] methionyl of fragment of human tPA (reteplase), under the control of a promoter operating in the said bacterial cell.

EFFECT: invention enables to obtain a recombinant fragment of human tissue plasminogen activator with a high yield.

10 cl, 6 dwg, 1 tbl, 8 ex

Description

Technical field

The invention relates to the field of biotechnology, and in particular to a technology for the production of biologically active substances (BAS) by genetic engineering, more specifically to methods for producing a human tissue-type plasminogen activator, corresponding to a recombinant human deletion variant of tissue plasminogen activator (TAP) reteplase, in therapeutically sufficient quantities.

The prior art.

Plasminogen is a glycoprotein synthesized in the liver and constantly circulating in the plasma, able to bind to thrombus fibrin. After enzymatic conversion (activation) from inactive plasminogen, plasmin is formed - a serine protease that breaks down fibrin, fibrinogen, as well as V, VIII and XII blood coagulation factors, reduces platelet adhesion and causes their disaggregation. The ability to activate plasminogen is possessed by endogenous (tissue plasminogen activator, urokinase or tissue plasminogen activator urokinase type, factor XII, kallikrein, kinins) and exogenous factors (bacterial streptokinase and staphylokinase).

Modern fibrinolytic (thrombolytic) drugs are based on stimulating the formation of plasmin from endogenous plasminogen. In this case, the systemic effect of the drug will be combined with the local one, that is, the drug will achieve the greatest activity on the surface of the thrombus, where plasminogen associated with fibrin is already present. Thrombolytics - plasminogen activators include bacterial enzyme preparations (streptokinase, anistreplase, staphylokinase), urokinase preparations, and tissue plasminogen activator preparations, TAPs (recombinant full-length TAPs and deletion variants of TAPs), as well as other, less common agents.

A tissue-type plasminogen activator or tissue plasminogen activator (tPA, TAP) is a serine protease (code for the International Enzyme Classifier EC 3.4.21.68), which catalyzes the conversion of an inactive plasminogen proenzyme to an active plasmin enzyme and is an important component of the fibrinolysis system. TAP is synthesized in vivo as a single-chain polypeptide (72 kDa), which is cleaved by various proteinases, including plasmin, tissue kallikrein and activated factor X, which hydrolyze the Arg275-Ile276 peptide bond to form chain A and chain B held together by a disulfide bond. The N-terminal region (A- or heavy chain) consists of several domains: residues 4-50 are homologous to the finger-like F-domain of fibronectin, which provides binding to fibrin; residues 50-87 form an E-domain homologous to epidermal growth factor; residues 87-176 (kringle 1) and 176-262 (kringle 2) are homologous to the kringle domains of plasminogen. The P domain, including residues 276-527 (B or heavy chain), is homologous to serine proteinases and contains a catalytic site of the active center, including His322, Asp371 and Ser478 (J Thromb Haemost. 2004 Apr; 2 (4): 541-6. Tissue-type plasminogen activator: a historical perspective and personal account. Collen D, Lijnen HR.). Separate TAP domains cause various enzyme functions, such as binding to fibrin, activation of plasminogen, binding to endothelial cell receptors, and rapid elimination from the body. The binding of TAP to the surface of fibrin is mediated by the region of the fibronectin finger and the region of the second kringle domain.

Endogenous TAP, determined in the blood, is an endothelial activator released into the bloodstream under the influence of various stimuli. The concentration of TAP in the blood is 6.6 ± 2.9 ng / ml. As a result of partial degradation of fibrin by plasmin, its binding to TAP increases. In the presence of fibrin, the catalytic efficiency (k _cat / K _M ) of TAP against plasminogen increases by a factor of 1,500.

There are several basic drugs based on recombinant human TAP (rhtAP) variants: alteplase (US Pat. No. 5,869,314), reteplase (European Patent Application EP0242836A1), TNKase (US Pat. No. 5,385,732 and 5,270,198). The concentration of natural TAP in plasma is very low and its isolation from donor plasma is economically disadvantageous.

Reteplase is a drug based on a non-glycosylated recombinant deletion variant of a human tissue type plasminogen activator. The recombinant polypeptide, weighing 39571 Da, includes 355 of 527 amino acids of the natural analogue, is produced in the Escherichia coli system (as a result of which it is non-glycosylated), contains the catalytic and Kringle 2 domains of the human tissue-type plasminogen activator, and does not contain the Kringle 1, ether, and fibronectin domains.

Structures that enhance the affinity of natural TAP to fibrin (fibronectin finger) and to liver cell receptors providing accelerated clearance (kringle 1, domain E, polysaccharide residues) are absent in the recombinant polypeptide, as a result of which the reteplase half-life is increased (compared to the full-size analog ) up to 13-16 minutes. The increase in half-life allows the clinic to use single or double intravenous administration of the drug instead of continuous infusion (JAMA. 2001 Jul 25; 286 (4): 442-9. Bolus fibrinolytic therapy in acute myocardial infarction. Llevadot J, Giugliano RP, Antman EM) . In turn, a decrease in the affinity for fibrin allows the free (non-fibrin-bound) drug to penetrate deep into the thrombus, which accelerates thrombolysis compared to full-size TAP. A number of studies indicate a comparatively higher reteplase efficacy compared to the full-size TAP (N Engl J Med. 1997 Oct 16; 337 (16): 1118-23. A comparison of reteplase with alteplase for acute myocardial infarction. The Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO III) Investigators) (Am J Health Syst Pharm. 1997 Nov 15; 54 Suppl 1: S27-30. Clinical trials in thrombolytic therapy. Part 2: The open-artery hypothesis and RAPID-1 and RAPID- 2).

Thus, a comparative study of the thrombolytic activity of reteplase and full-size TAP (alteplase) in an in vitro model showed that the full-size TAP remains mostly on the surface of the fibrin clot after washing, while deletion TAP penetrates deep into the clot, activating the plasminogen associated with fibrin contained in the clot. In a number of model studies in dogs with coronary artery thrombosis, it was shown that reteplase shows maximum efficiency compared with streptokinase, urokinase, anistreplaza, providing a minimum time for reperfusion (restoration of blood flow) of the coronary artery.

Clinical trials in which the comparative efficacy of a reteplase and a drug based on full-length TAP in acute myocardial infarction was studied showed that when using reteplase, the rate of reperfusion of the coronary arteries is significantly higher and the proportion of patients requiring subsequent angioplasty is lower. In a comparative clinical study of RAPID 2, during which angiography of the blood vessels of the heart was performed using a blind method after the administration of reteplase or full-length TAP, it was found that reteplase causes a more rapid restoration of blood flow in the affected coronary artery. It was also shown that the proportion of patients requiring additional interventions during the first 6 hours after starting therapy in case of using reteplase is half as much as when using full-size TAP (13.6% versus 25.6%, p = 0.004) (Lopez, LM, Am J. Health Syst. Pharm. 1997 Nov 15; 54 Suppl 1: S27-30. Clinical trials in thrombolytic therapy. Part 2: The open-artery hypothesis and RAPID-1 and RAPID-2).

Despite excellent clinical performance, reteplase currently occupies a limited global market share. The main technological problem in the production of reteplase is the combination of an ineffective producer strain based on the use of natural cDNA of the TAP fragment and an auxiliary plasmid with rare codon tRNA genes, as well as a very expensive refolding procedure consisting of the conversion of the crude reduced form of reteplase to mixed disulfide using large amounts of oxidized glutathione and subsequent protein renaturation in a very dilute solution in the presence of high concentrations of a ginina (European Patent Application ER0242836A1). A consequence of the use of these production technologies is the need to use expensive affinity purification of the renatured protein and a very low yield of the finished product.

A brief description of the present invention

The technical problem solved by the authors was the creation of a technology for rchTAP with a higher yield.

The technical result was achieved by creating a technology that includes a new expression plasmid DNA p10E-tPA encoding a fragment of a tissue plasminogen activator of a person, creating a producer strain E. coli based on it, and rhTAP isolation technology.

This solution is based on the 6388 bp plasmid DNA p10E-tPA developed by the authors, which encodes a 23 amino acid cleavable N-terminal leader peptide, which includes a decagystidine cluster and an enterokinase recognition sequence, and a sequence encoding a fragment of tissue activator fused with it human plasminogen. The indicated fragment contains codons optimal for E. coli, which allow increasing the expression level of the heterologous protein due to the effective translation of all amino acids of the polypeptide. The presence of the leader peptide allows purification of the polypeptide under denaturing conditions and refolding it not in solution, but on the surface of the metal chelate sorbent, which leads to a significant increase in the yield of the purified active target protein.

The aim of the present invention is the provision of an expression plasmid containing a DNA fragment encoding a fragment of a tissue plasminogen activator of a person, comprising a sequence encoding a cleavable N-terminal leader peptide comprising a decagidine cluster and an enterokinase recognition sequence fused in frame with a sequence encoding a fragment of a tissue plasminogen activator , under the control of a promoter functioning in a bacterial cell.

It is also an object of the present invention to provide the expression plasmid described above, wherein said plasmid is selected from the group consisting of p10E-tPA and pM-tPA plasmids.

It is also an object of the present invention to provide a bacterium belonging to the genus Escherichia transformed with the plasmid described above, a producer of the precursor of a recombinant fragment of a tissue plasminogen activator in humans.

It is also an object of the present invention to provide a bacterium as described above, wherein said bacterium is represented by E. coli BL21 [DE3] / p10E-tPA and E. coli BL21 [DE3] / pM-tPA strains.

It is also an object of the present invention to provide a precursor for a recombinant fragment of a human plasminogen tissue activator containing a cleavable N-terminal leader peptide comprising a decagystidine cluster and an enterokinase recognition sequence.

It is also an object of the present invention to provide a method for producing a recombinant fragment of a tissue plasminogen activator of a person, including culturing the bacterium described above in a nutrient medium, isolating inclusion bodies, solubilizing the precursor protein, denaturing metal chelate chromatography, refolding the precursor protein, preparing the mature protein by treatment with enterokinase and Isolation of Mature Protein

It is also an object of the present invention to provide the method described above in which E. coli strain BL21 [DE3] / p10E-tPA or E. coli strain BL21 [DE3] / pM-tPA is cultured.

Detailed description of the present invention

To implement the present invention, the main technical problem was the creation of a method for producing a recombinant fragment of a tissue plasminogen activator using a bacterium transformed with an expression plasmid containing a DNA fragment encoding a precursor of a recombinant fragment of a tissue plasminogen activator of human, comprising a sequence encoding a cleavable N-terminal leader peptide comprising decystididine cluster and enterokinase recognition sequence, and with a sequence cast with it in a frame encoding a recombinant fragment of a tissue plasminogen activator of a person under the control of a promoter that functions in a bacterial cell.

The term "expression plasmid" means plasmid DNA containing all the necessary genetic elements for the expression of a gene embedded in it, for example, such as a promoter, terminator. A specific example of the genetic elements necessary for the expression of a precursor of a recombinant fragment of a tissue plasminogen activator of a person in the expression cassette of the present invention is the T7 bacteriophage RNA polymerase promoter, but is not limited to it.

A DNA fragment encoding a precursor of a recombinant fragment of a tissue plasminogen activator of the person according to the present invention is, for example, a synthetic gene encoding a precursor of a recombinant fragment of a tissue plasminogen activator of a person, comprising a sequence encoding a cleavable N-terminal leader peptide comprising a decagystidine cluster and an enterocene recognition sequence fused in frame with a sequence encoding a recombinant fragment of a tissue asset human plasminogen atom. The indicated DNA fragment can be obtained by PCR (see Example 1, Fig. 1). Also, this DNA fragment can be obtained using the cloning technology of Sloning BioTechnology, described in PCT application WO2005071077.

In order to ensure efficient translation of the cloned gene into E. coli, it is preferable that in the sequence encoding the precursor of the recombinant fragment of human tissue plasminogen activator all rare codons are replaced by synonymous frequent codons.

The sequence of the gene encoding the precursor of the recombinant fragment of tissue activator of human plasminogen according to the present invention, is presented in the Sequence Listing under the number SEQ ID NO: 1. The amino acid sequence of the recombinant human TAP precursor according to the present invention is presented in the List of sequences under the number SEQ ID NO: 2. The amino acid sequence of a mature recombinant fragment of a tissue plasminogen activator of a person is a sequence under the number SEQ ID NO: 2 without the first 23 amino acids.

DNA fragments that encode essentially the same protein can be obtained, for example, by modifying the nucleotide sequence of a DNA fragment (SEQ ID NO: 1) encoding a precursor of a recombinant fragment of a tissue plasminogen activator of a person, for example, by the method of site-directed mutagenesis, so that one or more amino acid residues at a particular site will be delegated, replaced, inserted or added. DNA fragments modified as described above can be obtained using traditional processing methods to obtain mutations. DNA fragments that encode essentially the same protein can be detected by expression of DNA fragments having the mutation described above in the corresponding cell and establishing the activity of the expressed product.

Tissue plasminogen activator (synonyms: tPA, TAP, tissue type plasminogen activator) is a serine protease (cipher of the International Classification of Enzymes EU 3.4.21.68), which catalyzes the conversion of inactive plasminogen proenzyme to the active plasmin enzyme.

TAP is synthesized in vivo as a single chain polypeptide (72 kDa), which is cleaved by proteinases, via the Arg275-Ile276 bond, to form chain A and chain B held together by a disulfide bond. Separate TAP domains determine enzyme functions, such as binding to fibrin, plasminogen activation, binding to endothelial cell receptors, and rapid elimination from the body. The binding of TAP to the surface of fibrin is mediated by the region of the fibronectin finger and the region of the second kringle domain.

The expressed recombinant polypeptide, includes 355 of 527 amino acids of the natural analogue, contains the catalytic and Kringle 2 domains of the natural human tissue type plasminogen activator (does not contain the Kringle 1, ether, and fibronectin finger domains).

Indicators of functional activity, in which it is believed that the obtained protein has TAP properties, are determined by its proteolytic activity (its ability to hydrolyze the plasminogen peptide bond). So, for example, human TAP activity can be detected, for example, by hydrolysis of the synthetic substrate T2943 by the method described in Example 7. It is believed that the protein variant has the properties of human TAP, provided that the activity of this option is not less than 1% of the activity of the native human TAP.

The expression plasmid according to the present invention contains a DNA fragment encoding a precursor of a recombinant human TAP fragment comprising a sequence encoding a cleavable N-terminal leader peptide comprising a decagidine cluster and an enterokinase recognition sequence fused in frame with a sequence encoding a human TAP fragment under the control of a promoter functioning in a bacterial cell.

As the recombinant plasmid of the present invention, various plasmids capable of expression in the recipient cell can be used, such as plasmids pBR322, pMW119, pUC19, pET22b, pET28b and the like, but the list of plasmids is not limited to them.

A specific implementation of the present invention is a plasmid p10EK-tPA, which consists of:

1) a 41 bp NheI-NcoI fragment, which is a synthetic adapter encoding a decahystidine cluster;

2) an NcoI-HindIII fragment of pET28a (+) vector with a length of 5246 bp containing the region of the beginning of replication of the plasmid pBR322, the Rop-organizing Rop protein gene, the replication initiation site of bacteriophage f1, the sequence encoding aminoglycoside-3'-phosphotransferase, RNA promoter -polymerase bacteriophage T7; transcription termination site; a sequence encoding a repressor of a lactose operon;

3) a 1101 bp NheI-HindIII fragment encoding a human TAP fragment and an enterokinase recognition sequence fused in frame.

The indicated plasmid contains unique recognition sites for restriction endonucleases: ClaI (1251), PciI (2142), ApaI (4040), NcoI (5070), NheI (5111), EcoRI (5708), HindIII (6212).

The structure of plasmid p10EK-tPA is shown in Fig. 2.

Another specific embodiment of the present invention is the plasmid pM-tPA, which consists of:

1) an NcoI-HindIII fragment of pET28a (+) vector with a length of 5246 bp containing the region of the beginning of replication of plasmid pBR322, the Rop-organizing Rop protein gene, the bacteriophage replication initiation site f1, the sequence encoding aminoglycoside-3'-phosphotransferase, RNA promoter -polymerase bacteriophage T7; transcription termination site; a sequence encoding a repressor of a lactose operon;

2) a 1076 bp PciI-HindIII fragment encoding a methionine residue and a human TAP fragment.

The indicated plasmid contains unique recognition sites for restriction endonucleases: ClaI (1251), PciI (2142), ApaI (4040), NcoI (5004), NheI (5045), EcoRI (5642), HindIII (6146).

The structure of plasmid pM-tPA is shown in Fig. 3.

Using the created plasmids, it is possible to transform a bacterial cell, preferably a bacterium belonging to the genus Escherichia, susceptible to such transformation by the indicated plasmids. The choice of a particular cell is not critical, since the methodology and methods of transformation are well known to those skilled in the art. Although the expression level of the precursor of a TAP fragment of a person may vary depending on the type of cell and culturing conditions of the obtained transformant, the fact of expression of the target protein will take place subject to successful transformation of the recipient cell.

"Transformation of a cell with a plasmid" means the introduction of a plasmid into a cell using methods well known to those skilled in the art. Transformation with this plasmid leads to the expression of the gene encoding the protein of the present invention and to the synthesis of the protein in the bacterial cell. Transformation methods include any standard methods known to one skilled in the art, 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, a “bacterial cell producing a precursor of a human TAP fragment” means a bacterial cell having the ability to produce and accumulate a precursor of a human TAP fragment of the present invention when the bacterial cell of the present invention is grown in said growth medium. As used herein, the term “bacterial cell producing the precursor of a human TAP fragment” also means a cell that is capable of accumulating a product of a precursor of a human TAP fragment in an amount of not less than 1 mg / L (or 1 μg / 10 ⁹ cells), more preferably not less than 40 mg / l. The specified precursor fragment of the TAP of a person accumulates in the specified cell, preferably in the form of inclusion bodies.

It is preferable to use a bacterium belonging to the genus Escherichia for transformation with a recombinant plasmid containing a DNA fragment encoding the precursor of a human TAP fragment.

The term "bacterium belonging to the genus Escherichia" may mean that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia, the bacterium Escherichia coli (E. coli) may be mentioned.

The range of bacteria belonging to the genus Escherichia is not limited in any way, however, for example, the bacteria described in 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 specific example of a recipient strain for producing a producer of a precursor of a human TAP fragment of the present invention is, but is not limited to, Escherichia coli BL21 strain [DE3].

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

Cultural and morphological features of the strain: gram-negative rods, form threads; on an agar medium — whitish large colonies with an uneven edge. The activity of the strain is determined by densitometry electrophoregram. The strain is stored under the following conditions: Lurie-Bertrand medium, 1% glucose, 10% glycerol. The strain propagates under the following conditions - Lurie-Bertrand medium, 1% glucose, kanamycin sulfate 30 μg / ml.

Genetic features of the strain. The genotype of the strain is F ^- ompT gal dcm lon hsdS _B (r _B ^- m _B ^- ) λ (DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])

Transformation of the Escherichia coli strain BL21 [DE3] with the p10E-tPA plasmid or pM-tPA plasmid yields the producer strain BL21 [DE3] / p10E-tPA or the producer strain BL21 [DE3] / pM-tPA, respectively, each of which provides synthesis of a recombinant protein precursor of a human TAP fragment in an amount of 30-70% of the total protein content of cells.

The Escherichia coli strain BL21 [DE3] / p10E-tPA encodes a 10E-tPA precursor protein consisting of the amino acid sequence of a human TAP fragment and a 23 amino acid fused N-terminal leader peptide containing a decagystidine cluster and an enterokinase cleavage site immediately before the first amino acid of the human TAP fragment.

The Escherichia coli strain BL21 [DE3] / pM-tPA encodes an M-tPA precursor protein consisting of the amino acid sequence [-1] of a human TAP methionyl fragment.

A method for producing a human TAP fragment according to the present invention includes culturing the bacterium described above in a nutrient medium suitable for growing said prokaryotic cells, inducing a promoter of the TAP fragment precursor gene, isolating inclusion bodies, solubilizing the precursor protein, denaturing metal chelate chromatography, protein refolding precursor preparation of a mature protein by treatment with enterokinase and isolation of the indicated mature protein of a recombinant TAP fragment skill.

Features of plasmids and the results of their practical application are shown in the following drawings.

In Fig. Figure 1 shows the assembly scheme for the synthetic gene for the human TAP fragment from oligonucleotide primers and the preparation scheme for expression plasmids p10E-tPA, pM-tPA. The following notation is used: "TPA (no SP, 527 aa)" - tissue plasminogen activator, without signal peptide and cleaved propeptides; "A", "B" - chains A and B, respectively; "FT1" is the finger-like domain of fibronectin ("fibronectin finger"); "EG" - a domain homologous to epidermal growth factor; "KD1" and "KD2" are the kringle domains 1 and 2 (homologous to the kringle domains of plasminogen); "S1" is a proteolytic domain homologous to serine proteinases; "RET1" and "RET2" are fragments that make up the reteplase. The first and last amino acid fragments are indicated in parentheses. The dashed line indicates the polymerase chain reaction, the solid line indicates the restriction and ligation of DNA fragments.

In Fig. 2 shows a map of the expression plasmid p10E-tPA. The following notation is used: "pBR322ori" - region of the beginning of replication of plasmid pBR322; "ROP" - gene of the RNA-organizing protein Rop; "F1 ori" is the site of initiation of replication of bacteriophage f1; "KanR2" is a sequence encoding aminoglycoside-3'-phosphotransferase, which provides bacterial resistance to kanamycin; "T7 prom" - promoter of RNA polymerase of the bacteriophage T7; "T7term" - transcription termination site; "LacI" is the sequence encoding the repressor of the lactose operon; “UE-tPA” - open reading frame (OPC) of the polypeptide of the precursor protein of the human TAP fragment, including the detachable N-terminal additional peptide “10E leader” and the human TAP fragment “tPA”; “EK-PRO” is the site encoding the enterokinase recognition site, “stop” is the block of stop codons. The arrows indicate the directions of gene transcription, the numbers of the first and last nucleotides of the fragments are indicated in parentheses. The recognition sites of restriction endonucleases are in italics, and the numbers of nucleotides at the cutting points are indicated in parentheses.

In Fig. 3 shows a map of the expression plasmid pM-tPA. Designations are used similarly to Fig. 2, as well as “M-tPA” - an open reading frame (OPC) of the polypeptide of the precursor protein [-1] of the methionyl fragment of human TAP.

In Fig. Figure 4 shows the electrophoregram of the total protein, fractions of soluble and insoluble protein of cells of the producer strain BL21 [DE3] / p10E-tPA upon induction. Shake flask cultivation, 2xYT medium, induction of 1 mM IPTG, 37 ° C, 20 hours. Legend: T - total protein after induction, S - fraction of soluble proteins, I - fraction of insoluble proteins (inclusion bodies). M is a molecular weight marker. The molecular weights of the marker bands are indicated in kDa. The position of the target protein is indicated by the arrow. Productivity: 300 mg / L, target protein fraction:> 60% (by gel densitometry).

In Fig. Figure 5 shows the electrophoregram of the eluate obtained by purification of the precursor protein of the recombinant human TAP fragment by metal chelate chromatography under denaturing conditions. Designations: E - eluate fraction with EDTA-Na solution; E * - eluate fraction with EDTA-Na solution with increased image contrast; M is a molecular weight marker. The molecular weights of the marker bands are indicated in kDa. The position of the target protein is indicated by the arrow. Purity:> 98% (by gel densitometry).

In Fig. 6 shows an electrophoregram of a recombinant fragment of human TAP after renaturation on a metal chelate column. Designations: 1 - non-reducing conditions; 2 - restoring conditions. 20% of the monomer by densitometry gel.

The present invention will be described in more detail below with reference to the following non-limiting Examples.

Example 1. Obtaining plasmid DNA pAL-TPA

For the amino acid sequence including 355 of 527 amino acids of the natural human TAP (amino acids 1-3 and 176-527) with the added 23-amino acid N-terminal leader peptide (SEQ ID NO: 3) containing the enterokinase recognition sequence, reverse translation was performed DNA nucleotide sequence. In this case, codons optimal for the expression of this gene in E. coli class B were used, and the structure of the gene was optimized for the secondary structure of mRNA, GC composition, and the absence of undesirable regulatory elements (for example, the absence of internal ribosome binding sites) and the absence of extended repetitions, palindromes.

The CAI (Codon Adaptation Index) index, which reflects the efficiency of gene expression in this organism, for the obtained sequence was 0.84, which is a good prognostic indicator for the industrial suitability of the producer strain obtained on its basis. The obtained nucleotide sequence is shown in SEQ ID NO: 4.

The synthetic TAP gene was collected from two fragments of tPA-Fl (SEQ ID NO: 5) and tPA-F2 (SEQ ID NO: 6), and then an epitope was added for immunochemical detection by PCR. For this, constructs pGEM-tPA-F1 and pAL-TPA-F2 containing fragments tPA-F1 and tPA-F2, respectively, were obtained, they were assembled in pGEM-tPA, PCR was performed, and pAL-tPA with a complete leader peptide was obtained.

The tPA-F1 fragment (SEQ ID NO: 5) was collected by polymerase chain reaction (PCR) with primers T1F1-T1F7 and T1R1-T1R7 (SEQ ID NO: 6-19), the sequences of which are shown in Table 1.

PCR was performed on a Tertsik MS2 instrument (DNA Technology, Russia). Preparative reactions were carried out in a volume of 50 μl. An incubation mixture of the following composition was prepared: 1x buffer for thermostable DNA polymerase; 10 pM of each primer oligonucleotide; 2 mM each deoxyribonucleotide triphosphate; 2 units thermostable DNA polymerase. 50 μl of mineral oil was layered on top of this mixture and amplified according to the scheme: 1 cycle - denaturation - 94 ° C, 3 min; 25 cycles - denaturation 94 ° C, 30 sec; annealing 62 ° C, 30 sec; chain extension 72 ° C, 120 sec; 1 cycle - chain extension 72 ° С, 10 min.

PCR products were isolated from 1% agarose gel using the Wizard SV Gel and PCR Clean-Up System reagent kit (Promega, USA) according to the manufacturer's protocol, and then they were ligated into the pGEM-T T-vector (Promega, USA ) using T4 phage DNA ligase and standard buffer solution (Fermentas, Lithuania). Ligation was carried out in a volume of 10 μl with a molar ratio of vector and insert 1:10, for 2-20 hours at room temperature. The obtained ligase mixtures transformed E. coli cells of strain DH5α, with the genotype F-φ80lacZΔM15 Δ (lacZYA-argF) U169 recA1 endA1 hsdR17 (r _k -, m _k +) phoA supE44 λ-thi-1 gyrA96 relA1. For this, 5 μl of the ligase mixture was added to 200 μl of a frozen suspension of E. coli cells, incubated on ice for 20 minutes to sorb plasmid DNA, heated to 42 ° C for 45 seconds, and incubated on ice for 5 minutes. Then 800 μl of LB nutrient broth was added and incubated at 37 ° C for 60 min. After incubation, the suspension was transferred to a Petri dish with a solid agar medium containing ampicillin at a concentration of 100 μg / 1 ml of agar and placed in a thermostat for 18 hours at 37 ° C.

E. coli colonies selected as a result of blue-white screening were analyzed by clone PCR using primers for the recipient plasmid sequences: M13for (SEQ ID NO: 20) and M13rev (SEQ ID NO: 21). Selected clones were grown in 5 ml LB nutrient broth and plasmid DNA was isolated using the Wizard Plus SV Minipreps reagent kit (Promega, USA) according to the manufacturer's protocol, the primary nucleotide sequence of the pGEM-tPA-F1 construct was confirmed by PCR sequencing using primers T7prom (SEQ ID NO: 22) and SP6 (SEQ ID NO: 23).

The tPA-F2 fragment (SEQ ID NO: 6) was collected by PCR from T2F1-T2F6 and T2R1-T2R6 primers (SEQ ID NO: 24-35), the sequences of which are shown in Table 1. PCR was performed as in the preparation of the tPA fragment -F1. The PCR product was cloned into the pAL-TA T-vector (Eurogen, Russia), blue-white screening was performed, and colony PCR was performed using standard primers for the vector sequences M13for (SEQ ID NO: 20) and M13rev (SEQ ID NO: 21 ), positive clones were expanded in 2xYT-Amp liquid medium, plasmid DNA was isolated using the Gene JET Plasmid Miniprep Kit (Fermentas, Lithuania), the nucleotide sequence of the pAL-TA-TPAF2 insertion region was determined by PCR sequencing using primers T7prom (SEQ ID NO: 22) and SP6 (SEQ ID NO: 23).

Obtaining the construction of pGEM-tPA (assembly of fragments of the TAP gene)

Plasmid DNA pGEM-tPA-F1, containing the tPA-F1 fragment in direct orientation, was digested with EcoRI endonuclease (Permentas, Lithuania), dephosphorylated using alkaline phosphatase (Fermentas, Lithuania); pAL-TA-TPAF2 plasmid DNA was digested with excess EcoRI endonuclease. The restriction products of plasmids pGEM-tPA-F1 and pAL-TA-TPAF2 were subjected to separation in a 1% agarose gel, isolated using the WizardSV Gel and PCR Clean-Up System kit (Promega, USA). The isolated fragments were ligated using T-4 DNA ligase (Fermentas, Lithuania) according to the manufacturer's procedure. Colony PCR was analyzed using standard primers M13for (SEQ ID NO: 20) and M13rev (SEQ ID NO: 21), clones containing the insert were expanded in 2xYT-Amp liquid medium, plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit (Fermentas, Lithuania), confirmed the nucleotide sequence of the pAL-TA-TPAP2 insert region by PCR sequencing with primers T7prom (SEQ ID NO: 22) and SP6 (SEQ ID NO: 23).

Introduction of additional amino acids and a second stop codon into the open reading frame of rhtAP

Conducted 15 cycles of PCR (according to the scheme: denaturation - 94 ° C, 3 min; 15 cycles - denaturation 95 ° C, 15 sec, annealing 55 ° C, 25 sec, chain extension 72 ° C, 90 sec; final chain extension 72 ° C, 10 min) using specific primers AD-TPA-2stop-r (SEQ ID NO: 36) and AD-TPA-DYKin-f (SEQ ID NO: 37) taken in an amount of 10 pM each and 20 ng pGEM -tPA used as a matrix. PCR products were separated on a 1% agarose gel, the desired fragment was cut from the gel, and DNA was isolated using the Wizard SV Gel and PCR Clean-Up System reagents (Promega, CLUA) according to the manufacturer's protocol. The purified products were ligated into the pAL-TA vector plasmid (Evrogen, Russia) using T4 phage DNA ligase and standard buffer solution (Fermentas, Lithuania). Ligation was carried out in a volume of 20 μl, with a molar ratio of vector and insert of 1:10, for 10 hours at room temperature. The obtained ligase mixtures transformed E. coli cells of strain DH5α. E. coli colonies selected as a result of blue-white screening were analyzed by clone PCR using M13for primers (SEQ ID NO: 20) and M13rev (SEQ ID NO: 21). Selected clones were grown in 5 ml LB nutrient broth and plasmid DNA was isolated using the Wizard Plus SV Minipreps kit (Promega, USA) according to the manufacturer's protocol. The resulting pAL-tPA genetic constructs were analyzed by PCR sequencing with primers M13for (SEQ ID NO: 20) and M13rev (SEQ ID NO: 21).

After that, preparative restriction of the plasmid pAL-tPA with restriction endonucleases NheI and HindIII was performed. The reaction products were separated on a 1% agarose gel and isolated using the Wizard SV Gel & PCR Clean-Up System no. Reagent kit according to the manufacturer's method of the donor fragment to obtain the expression plasmid p10E-tPA.

Example 2. Obtaining a vector plasmid p10E

The sequence encoding a fragment of the N-terminal leader peptide comprising the decagystidine cluster was obtained by annealing two partially complementary oligonucleotides AD-10H-NcoF (SEQ ID NO: 38) and AD-10H-NheR (SEQ ID NO: 39) with the formation of protruding " sticky 5'-ends. For this, 100 pm of each oligonucleotide was introduced into the test tube, heated to 95 ° C, and slowly cooled to room temperature.

The recipient plasmid pET28a (+) (Novagen) was sequentially digested with each of the NcoI and NheI endonucleases. First, aliquots of the plasmid were incubated for 2 hours at 37 ° C with each of the restriction enzymes, then the complete linearization of the plasmids was controlled by agarose gel electrophoresis (within the sensitivity of the method), after which a second restriction enzyme was added and incubated for another 2 hours. Then the samples were combined, the restriction enzymes were inactivated by heating at 65 ° C for 20 minutes. The restricted plasmid was reprecipitated with 3 volumes of ethanol, centrifuged for 10 minutes at a speed of 13200 rpm at room temperature, the precipitate was washed with 70% alcohol, dissolved in water, and used to set up a ligation reaction at a working concentration of 10 ^-2 μg / μl. The isolated fragments were ligated using T-4 DNA ligase (Fermentas, Lithuania) according to the manufacturer's procedure. After that, cells of E. coli strain DH5α were transformed, as described above. E. coli colonies were analyzed by clone PCR using AD-10H-NcoF oligonucleotides (SEQ ID NO: 38) and T7t (SEQ ID NO: 40). Selected clones were grown in 5 ml of 2xYT-Kan nutrient broth, plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit (Fermentas, Lithuania) according to the manufacturer's protocol. For the obtained p10E genetic constructs, the DNA nucleotide sequence was determined by PCR sequencing with T7t primer (SEQ ID NO: 40).

Example 3. Obtaining expression plasmid DNA p10E-tPA

The recipient plasmid p10E was sequentially digested with each of the NheI and HindIII endonucleases. First, aliquots of the plasmid were incubated at 37 ° C for 2 hours with each of the restriction enzymes, the linearization of the plasmids was monitored by agarose gel electrophoresis, a second restriction enzyme was added, and incubated for another 2 hours. Then the samples were combined, the enzymes were inactivated by heating at 65 ° C for 20 minutes, and alkaline phosphatase dephosphorylation (Fermentas, Lithuania) was performed according to the manufacturer's protocol. Alkaline phosphatase was inactivated by heating to 85 ° C for 20 minutes. The digested and dephosphorylated plasmid was reprecipitated with 3 volumes of ethanol, centrifuged for 10 minutes at a speed of 13,200 rpm at room temperature, the precipitate was washed with 70% alcohol, dissolved in water and used to set up a ligation reaction at a working concentration of 10 ^-2 μg / μl.

The ligation reaction of the purified fragment corresponding to the tPA minigene and the recipient plasmid was carried out as described above. After that, cells of E. coli strain DH5α were transformed, as described above.

E. coli colonies were analyzed by PCR from clones from primers T7prom (SEQ ID NO: 22) and T7t (SEQ ID NO: 40). Selected clones were grown in 5 ml of 2xYT-Kan nutrient broth, plasmid DNA was isolated with the Wizard Plus SV Minipreps kit (Promega, USA) according to the manufacturer's protocol. Using PCR sequencing for the p10E-tPA construct, the nucleotide sequence of both complementary DNA strands was determined for the insertion region using primers T7prom (SEQ ID NO: 22) and T7t (SEQ ID NO: 40). As a result of sequencing, it was found that the obtained plasmid preparation does not contain mutations in the insertion region, i.e., the correct sequence of the TAP gene is encoded. The structure of the plasmid is shown in Fig.2.

Example 4. Obtaining plasmid DNA pM-tPA

Conducted 15 cycles of PCR (according to the scheme: denaturation 94 ° С, 3 min; 15 cycles of denaturation 95 ° С, 15 sec; chain extension 55 ° С, 25 sec; extension 72 ° С, 90 sec; final chain extension 72 ° С, 10 min) using specific primers AD-TPAM-PciF (SEQ ID NO: 41) and AD-TPA-2stop-r (SEQ ID NO: 36) taken in an amount of 10 pM each and 20 ng pAL-tPA used as a matrix. PCR products were separated on a 1% agarose gel, the desired fragment was cut from the gel, and its selection was performed using the Wizard SV Gel and PCR Clean-Up System kit (Promega, USA). The purified products were ligated into the pAL-TA vector plasmid (Evrogen, Russia) using T4 phage DNA ligase and standard buffer solution (Fermentas, Lithuania). Ligation was carried out in a volume of 20 μl at a molar ratio of vector and insert of 1:10, for 18 hours at room temperature, and then E. coli strain DH5α cells were transformed with ligase mixtures. E. coli colonies selected as a result of blue-white screening were analyzed by PCR from clones from primers M13for (SEQ ID NO: 20) and M13rev (SEQ ID NO: 21). Selected clones were grown in 5 ml of 2xYT-Amp nutrient broth and plasmid DNA was isolated from them using the Wizard Plus SV Minipreps kit (Promega, USA). The resulting pAL-M-tPA genetic constructs were analyzed by PCR sequencing with the primers M13for (SEQ ID NO: 20) and M13rev (SEQ ID NO: 21).

After this, preparative restriction of the plasmid pAL-M-tPA with restriction endonucleases PciI and HindIII was performed. The reaction products were separated on a 1% agarose gel and isolated using the Wizard SV Gel & PCR Clean-Up System reagent kit.

The recipient plasmid p10E was sequentially digested with each of the NcoI and HindIII endonucleases; for this, aliquots of the plasmid were incubated for 2 hours at 37 ° C with each of the restriction enzymes, linearization of the plasmids was controlled by agarose gel electrophoresis, a second restriction enzyme was added, and incubated for another 2 hours. Then the samples were combined, the enzymes were inactivated by heating at 65 ° C for 20 minutes, and alkaline phosphatase dephosphorylation (Fermentas, Lithuania) was performed according to the manufacturer's protocol. Alkaline phosphatase was inactivated by heating to 85 ° C for 20 minutes. The digested and dephosphorylated plasmid was reprecipitated with 3 volumes of ethanol, centrifuged for 10 minutes at a speed of 13,200 rpm at room temperature, the precipitate was washed with 70% alcohol, dissolved in water and used to set up a ligation reaction at a working concentration of 10 ^-2 μg / μl. The ligation reaction of the purified fragment corresponding to the M-tPA minigene and the recipient plasmid was carried out as described above. After that, cells of E. coli strain DH5α were transformed. E. coli colonies were analyzed by PCR from clones from primers T7prom (SEQ ID NO: 22) and T7t (SEQ ID NO: 40). Selected clones were grown in 5 ml of 2xYT-Kan nutrient broth, plasmid DNA was isolated from them using the Wizard Plus SV Minipreps kit. Using PCR sequencing for the pM-tPA construct, the nucleotide sequence of both complementary DNA strands was determined for the insertion region using primers T7prom (SEQ ID NO: 22) and T7t (SEQ ID NO: 40). As a result of sequencing, it was found that the obtained preparation of plasmid pM-tPA does not contain mutations in the insertion region, that is, the correct sequence of the [-1] M-TAP gene is encoded. The structure of the plasmid is shown in Fig. 3.

Example 5. Obtaining producer strains of E. coli BL21 [DE3] / p10E-tPA and E. coli BL21 [DE3] / pM-tPA, evaluating the productivity of producer strains and localization of the target protein

To obtain a producer strain of the recombinant TAP fragment, the constructs obtained in Examples 3 and 4 were used to transform competent cells of Escherichia coli BL21 (DE3) (with the genotype F-ompT hsdS _B (rm-) gal dcm (DE3)) and clones were selected preserving the level of biosynthesis of the recombinant polypeptide after induction of at least 30% of the total cellular protein for at least four consecutive passages.

To obtain the E. coli strain BL21 [DE3] / p10E-tPA, the producer of the precursor protein of the human TAP fragment, cells of the E. coli BL21 [DE3] strain were transformed with the expression plasmid p10E-tPA.

To obtain the E. coli strain BL21 [DE3] / pM-tPA, the producer [-1] of the methionyl fragment of human TAP, cells of the E. coli strain BL21 [DE3] were transformed with the expression plasmid pM-tPA.

E. coli BL21 [DE3] transformants were plated on 2xYT agar medium supplemented with kanamycin up to 30 μg / ml and glucose up to 2%, and the target proteins were analytically expressed for five randomly selected clones of a typical phenotype. Clones were grown in nutrient broth with the addition of kanamycin up to 30 μg / ml and glucose solution up to 2% for 6-7 hours, a new portion of the nutrient medium was inoculated with a ratio of 1: 100, the culture was grown until the optical density reached 2 O.E., induced isopropylthio-β-D-galactoside (IPTG) and cultured for another 2 hours. After cultivation, the cell pellet was separated by centrifugation, the cells were resuspended in a solution of 10 mM Tris-HCl, 2 mM EDTA-Na, 0.1% Triton-X100, 10 μg / ml lysozyme in the ratio of 10 ml of solution per 1 g of cell paste, the suspension was kept 30 min on ice and cell destruction was performed with an ultrasonic dispersant until the apparent viscosity of the suspension disappeared. Samples were taken for electrophoretic analysis, the soluble and insoluble fraction of proteins was separated by centrifugation in a microcentrifuge, the precipitate was additionally resuspended in the same solution, and precipitated by centrifugation. The results of electrophoretic analysis of total protein and fractions of soluble and insoluble proteins for strain BL21 [DE3] / p10E-tPA are shown in Fig. 4.

The electrophoretic mobility of both target proteins corresponds to the calculated values; the intensity of the target protein band for both target proteins is similar. According to gel electrophoresis of protein fractions, both target proteins were almost completely localized in the insoluble fraction of proteins, i.e. were in the form of “inclusion bodies”.

Example 6. Isolation, purification and renaturation of recombinant TAP

The producer strain BL21 [DE3] / p10E-tPA was seeded from the museum with a loop by a draining stroke on a Petri dish with agarized LB medium containing 30 μg / ml kanamycin and 1% glucose, grown for 14 hours at 37 ° C. One single colony of the strain was transferred to 5 ml of LB liquid medium containing 30 μg / ml kanamycin and 1% glucose and grown on a shaker for 14 hours at 37 ° C. The contents were inoculated with 250 ml of 2xYT medium containing 30 μg / ml kanamycin and 0.1% glucose, grown on a shaker for 3.5 hours at 37 ° C, bacterial suspension samples were taken for analysis, IPTG was added to a final concentration of 1 mM and grown for another 3-15 hours

The cell pellet was separated by centrifugation, resuspended in 20 ml of solution A (50 mM Tris pH = 7.4, 2 mM EDTA), lysozyme up to 0.1 mg / ml and Triton X100 up to 0.1% were added, incubated for 30 min on ice. Cells and genomic DNA were destroyed using an ultrasonic disperser in 10 s pulses until the increased viscosity of the suspension disappeared. The precipitate was separated by centrifugation for 10 minutes at 20,000 rpm. The precipitate was resuspended in solution A, NP-40 detergent was added to 1%, the suspension was treated with an ultrasonic disperser until the sediment particles larger than 1 mm disappeared, and the precipitate was separated by centrifugation for 10 min at 20,000 rpm. The resulting precipitate was resuspended in solution A, NaCl was added to 500 mM, the suspension was treated with an ultrasonic disperser until the sediment particles larger than 1 mm disappeared, and the precipitate was separated by centrifugation for 10 min at 20,000 rpm. The obtained precipitate was resuspended in a solution of 50 mM Tris pH 7.4, the suspension was treated with an ultrasonic disperser until the sediment particles larger than 1 mm disappeared, and the precipitate was separated by centrifugation for 10 min at 20,000 rpm. The resulting precipitate of enriched inclusion bodies was stored at -70 ° C.

To solubilize the target protein, solution B (6 M guanidine chloride, 50 mM sodium phosphate, 50 mM beta-mercaptoethanol, pH 8.0) was added to the precipitate of inclusion bodies in a ratio of 10 ml of solution per 1 g of precipitate. The suspension was incubated with stirring for 2 hours at 37 ° C, insoluble cell debris was separated by centrifugation for 10 min at 20,000 rpm. The supernatant was diluted 5 times with solution B (6 M urea, 50 mm sodium phosphate, 300 mm sodium chloride, pH 8.0) to reduce the final concentration of beta-mercaptoethanol, the precipitate was separated by centrifugation for 10 min at 20,000 rpm and the supernatant was applied on a column with a Chelating Sepharose Fast Flow sorbent (GE Healthcare, CHIA) containing nickel chelated ions and balanced solution B. The column was washed sequentially with solution G (6 M urea, 50 mm sodium phosphate, 300 mm sodium chloride, pH 7.0) and solution D (6 M urea, 50 mm sodium phosphate, 300 mm sodium chloride Ia, 50 mM imidazole, pH 7.0) to stabilize the baseline. The purified protein was eluted with solution E (6 M urea, 50 mM sodium phosphate, 300 mM sodium chloride, 50 mM EDTA-Na, pH 7.0). The eluate was concentrated by ultrafiltration to a final protein concentration of 20 mg / ml and desalted by diafiltration, and the resulting solution was stored frozen. The polypeptide was purified under denaturing conditions to apparent homogeneity (more than 95% purity by densitometry of the electrophoregram, Fig. 5).

For refolding in solution, DTT was added to the thawed protein to a concentration of 10 mM and incubated at 37 ° C for 2 hours. The protein was then diluted with refolding buffer solution (150 mM TRIS, pH 8.5, 0.5 M arginine, 2 mM EDTA, 0.2 mM reduced glutathione, 0.05% Tween-80 and 2.25 M urea) to a concentration 7 μg / ml and incubated at room temperature for three days. After this, the protein was again concentrated to 2 mg per ml. The resulting preparation possessed 50-90% specific proteolytic activity in comparison with the reteplase standard.

The refolding of the M-tPA precursor protein was carried out similarly to the procedure described above, but metal chelate chromatography was not performed under denaturing conditions, but the reconstituted denatured protein was introduced into the refolding directly from the composition of inclusion bodies. The specific proteolytic activity of 10E-tPA and M-tPA did not have significant differences.

The authenticity of the 10E-tPA precursor protein was studied by SELDI mass spectrometry. For this, an aliquot of the renatured protein was subjected to reduction of disulfide bonds by dithiothreitol, blocking of the free cysteine residues by iodoacetamide and applied to the surface of the H-50 chip (Ciphergen, United States), the proteins were adsorbed, and the chip slots were washed according to the manufacturer's instructions and a saturated matrix solution was applied to the chip slots. The mass spectrum of the sample was recorded in a ProteinChip Reader II device (Ciphergen, USA). A dominant molecular weight of 43,497 Da was recorded with a significant peak width and asymmetry. The theoretically calculated molecular weight of 10E-tPA protein, taking into account the chemical modifications, is 43344 Da and is in satisfactory agreement with the experimentally found one - the determination error is 3.5%.

Example 7. Purification and renaturation of immobilized 10E-tPA

Column refolding was performed according to the general scheme of Fan et. al. (J. Biophis. Biochem. Meth., 2008) as amended.

1. Inclusion bodies were dissolved in 8M guanidine hydrochloride, 50 mM Tris pH 7.5 and 50 mM beta-mercaptoethanol in a ratio of 10 ml of solution per 1 g of Taurus. The resulting solution was diluted 5 times with solution A (8 M urea, 0.5 M NaCl, 50 mm Tris pH 7.5) and applied to a column with a Ni-NTA sorbent (Qiagen), balanced with the same solution, in a ratio of 20 mg Taurus per 1 ml of sorbent.

2. The column was washed with 5 volumes of solution A and 5 volumes of solution A, additionally containing 0.5 M arginine hydrochloride.

3. A linear gradient was carried out over 10 column volumes from solution A (8 M urea, 0.5 M NaCl, 50 mM TRIS pH 7.5) to solution B (100 mM TRIS pH 8.5, 0.5 M arginine, 0.05% Tween 80).

4. The target protein was eluted with a solution of 0.5 M arginine, 2 M urea, 0.05% Tween 80, 50 mM EDTA, 50 mM TRIS pH 8.5, 0.5 M NaCl.

An analysis of the eluted renatured 10E-tPA was performed using SDS-PAGE under non-reducing conditions (Fig. 6). The purity of the target protein after purification was> 90% by densitometry of electrophoregrams. A typical yield of purified and renatured 10E-tPA was 30-50%. The proteolytic activity of 10E-tPA was measured by hydrolysis of a synthetic substrate T2943 (CH3SO2-D-HHT-Gly-Arg-pNA · AcOH, where NNT is hexahydrotyrosine, pNA is p-nitroanilide, manufactured by Sigma-Aldrich, USA) according to the method of the substrate manufacturer. The specific proteolytic activity of the renatured 10E-tPA monomer was at least 10% of the reteplase standard.

Example 8. Separation of the leader peptide and purification of the mature tPA protein

Separation of the N-terminal peptide was carried out using recombinant enterokinase (Sigma, United States) using an enzyme: substrate ratio of 1:10 (by weight). The reaction was conducted for 2 hours at 37 ° C in the presence of 5 mM calcium chloride. Enterokinase, unreacted 10E-tPA precursor protein, free 10E peptide and residual impurity proteins were separated by passing the reaction mixture through a column filled with Chelating Sepharose FF sorbent (GE Healthcare, USA) with immobilized cobalt ions. The obtained completely purified solution of the mature tPA protein located in the breakthrough fraction from the column was concentrated and desalted by ultrafiltration and stored at a temperature of -20 ° C. The specific proteolytic activity of the mature tPA protein and the 10E-tPA precursor protein did not differ significantly.

The results showed that the inclusion in the sequence of the synthesized protein of a short additional N-terminal peptide encoded by codons optimal for E. coli allows chromatographic purification of the expressed protein under denaturing conditions by metal chelate chromatography and to combine the immobilized protein refolding procedure with this stage of purification, which, in turn, allows you to get a fully purified and renatured product using a single chromatographic stage. If necessary, this peptide can be removed by treatment with enterokinase and subsequent repeated metal chelate chromatography.

The advantages of the proposed E. coli strain BL21 [DE3] are the use of bacteria with the Lon OmpT phenotype, which excludes the possibility of proteolytic cleavage of the synthesized de novo recombinant TAP and contamination of the secreted protein with the most active E. coli proteases. The T7 bacteriophage R7 polymerase RNA polymerase gene integrated in the genome of the recipient strain under the control of the lacUV5 promoter using the T7-1ac promoter and T7 terminator in plasmids leads to fast and efficient protein production.

Another common advantage of the used strain, expression vector and biosynthesis strategy is the ability to carry out induction without changing the temperature of cultivation.

Although the invention has been described in detail with reference to Examples, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention.

Claims (10)

1. Plasmid for expression in the cells of a bacterium of the genus Escherichia of the precursor of a recombinant fragment of human tissue plasminogen activator (TAP) (reteplase) containing a cleavable N-terminal peptide comprising a decagidine cluster and a fused recognition sequence of the enterokinase, or [-1] methionyl fragment Human TAP (reteplases), under the control of a promoter that functions in a bacterial cell, in the following sequence essentially containing:
- a fragment of the vector pET28a (+) containing the site for the initiation of replication of bacteriophage f1, the sequence encoding aminoglycoside-3'-phosphotransferase, the region of origin of replication of plasmid pBR322, the gene of the Rop-organizing protein Rop;
- a sequence encoding a repressor of a lactose operon;
- a DNA fragment containing a promoter that functions in a bacterial cell and a sequence encoding a methionine residue [-1], or a synthetic adapter that encodes a cleavable N-terminal leader, including a decagidine cluster and an enterokinase recognition sequence, fused in frame with the sequence encoding the precursor recombinant fragment of human TAP (reteplase);
- a DNA fragment encoding a precursor of a recombinant human TAP fragment (reteplase) or [-1] a methionyl fragment of human TAP (reteplase); and
- plot termination of transcription. 2. The plasmid according to claim 1, characterized in that said DNA fragment encoding the precursor of a recombinant human TAP fragment with a cleavable N-terminal peptide comprising a decagidine cluster and a fused enterokinase recognition sequence is a DNA fragment shown in the sequence listing under number SEQ ID NO: 1 or a variant thereof. 3. The plasmid according to claim 1, characterized in that the specified promoter is a promoter of RNA polymerase of the bacteriophage T7 4. The plasmid according to claim 1, characterized in that the plasmid is selected from the group consisting of plasmids p10E-tPA, pM-tPA. 5. The bacterium belonging to the genus Escherichia, transformed with the plasmid according to claim 1, is the producer of the precursor of the recombinant fragment of human TAP (reteplase) or [-1] methionyl fragment of human TAP (reteplase). 6. The bacterium according to claim 5, characterized in that said bacterium is selected from the group consisting of E. coli BL21 [DE3] / p10E-tPA, E. coli BL21 [DE3] / pM-tPA bacteria. 7. The precursor of a recombinant fragment of human TAP (reteplase) with a cleavable N-terminal peptide comprising a decagystidine cluster and a fused enterokinase recognition sequence obtained by culturing the bacterium according to claim 5 in a nutrient medium. 8. The precursor of the recombinant fragment of human TAP (reteplase) according to claim 7, containing the amino acid sequence presented in the List of sequences under the number SEQ ID NO: 2 or its functional variant. 9. The method for producing the precursor of the recombinant human TAP fragment (reteplase) according to claim 7, including culturing the bacterium according to claim 5 in a nutrient medium, isolating inclusion bodies, solubilizing the precursor protein, metal chelate chromatography under denaturing conditions, refolding the precursor protein, obtaining mature protein by treatment with enterokinase and isolation of the indicated mature human TAP fragment protein. 10. The method according to claim 9, characterized in that the E. coli strain BL21 [DE3] / p10E-tPA is cultured.

Images