RU2496877C2 - PLASMID VECTOR pHYP WITH HIGH SEGREGATION STABILITY FOR EXPRESSION OF RECOMBINANT PROTEIN, BACTERIUM - PRODUCENT OF PRECURSOR OF RECOMBINANT PROTEIN AND METHOD TO PRODUCE RECOMBINANT PROTEIN - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention is a vector for production of a vector for expression in a bacterial cell of a precursor of a target recombinant protein, fused with an N-end peptide, containing a decahistidine cluster and a site of recognition with proteinase, substantially containing of a section of initiation of replication of pBR322 ori; a gene of a repressor of a lactose operon; a bacterial promotor; an area coding the N-end leader peptide, containing a decahistidine cluster and, not necessarily, a site of recognition with proteinase; a section of cloning (polylinker); a section of termination of transcription functioning in the bacterial cell; a fragment coding a non-genome pair toxin-antitoxin, at the same time the gene of antitoxin is oriented so that the direction of its transcription matches the direction of transcription of the target gene; the gene coding the bacterial marker of selection. The invention also relates to a vector for expression in a bacterial cell of a precursor of a target recombinant protein fused with an N-end peptide, a bacterium containing such vector and the method for production of the recombinant protein using the bacterium.

EFFECT: invention makes it possible to produce a new vector with high segregation stability for highly efficient expression of recombinant proteins with a leader N-end peptide fused in the frame, containing a decahistidine cluster and a site of recognition with proteinase.

9 cl, 12 dwg, 1 tbl, 4 ex

Description

The scope of the invention.

The invention relates to the field of biotechnology and genetic engineering and is a plasmid vector with increased segregation stability for the expression of recombinant proteins with a frame-fused N-terminal leader peptide.

State of the art

Since modern industrial systems for the biosynthesis of heterologous proteins in bacterial cells involve the use of strong inducible promoters, a significant limitation of their productivity is the insufficient segregation stability of expression plasmids, i.e., there is a noticeable probability of a bacterial cell losing all copies of the plasmid during division. The loss of the expression plasmid by a part of the cells and the resulting decrease in the productivity of the expression system is especially noticeable during prolonged induction of the target gene and during cultivation of the producer strain with insufficient concentration (or complete absence) in the selective antibiotic medium.

To increase the segregation stability of E. coli plasmids, a number of vectors have been developed, including “positive selection” systems based on the disruption of the formation of the cytotoxic product upon insertion of the target fragment — MCS in the SacB region [Pierce, J. C., B. Sauer, et al. (1992). "A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy." Proc Natl Acad Sci USA 89 (6): 2056-2060], gpE phage ФХ174 [Henrich, B. and R. Plapp (1986). "Use of the lysis gene of bacteriophage phi X174 for the construction of a positive selection vector." Gene 42 (3): 345-349], a pKG2 plasmid carrying a genomic DNA digesting restriction enzyme under the control of the LacUV5 promoter [Kuhn, I., F. H. Stephenson, et al. (1986). "Positive-selection vectors utilizing lethality of the EcoRI endonuclease." Gene 42 (3): 253-263], CcdB and Kid [Bernard, P. and M. Couturier (1991). "The 41 carboxy-terminal residues of the miniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein." Mol Gen Genet 226 (1-2): 297-304; [Gabant, P., T. Van Reeth, et al. (2000). "New positive selection system based on the parD (kis / kid) system of the R1 plasmid." Biotechniques 28 (4): 784-788; U.S. Patents 6,180,407, 7,176,029, 5,910,438 (Universite Libre de Bruxelles)]. Such systems cannot be used in high-copy plasmids due to the inability to completely suppress the formation of a toxic product. In addition, plasmid vectors containing the above genes and genetic loci are suitable for transformation of only special, compatible with them, strains of E. coli and contain extended sections of actively transcribed non-targeted DNA, which reduce the productivity of the heterologous protein biosynthesis system.

Another, more suitable for industrial use, type of segregation stabilization systems of plasmids is a pair of transcribed genes encoded by a plasmid - a gene of a cell-toxic protein with stable mRNA and a specific antitoxin gene with unstable mRNA. General information about such toxin-antitoxin systems is provided by Cooper T, Paixao T, Heinemaim JA. (Within-host competition selects for plasmid-encoded toxin-antitoxin systems, Proc. R. Soc. On October 22, 2010 277: 3149-3155).

Among the toxin-antitoxin systems, the Hok / Sok genetic locus (also known as parB) of the natural E. coli R1 plasmid is of most practical interest.

Hok / Sok locus first described in (The parB (hok / sok) Locus of Plasmid R1: A General Purpose Plasmid Stabilization System. Nature Biotechnology 6, 1402-1405 (1988)); consists of the hok gene, which encodes a 52 amino acid toxin that causes dissipation of the membrane potential (the mechanism is similar to the action of bacteriophage lysis proteins - cholins (holin - from the hole “hole”), which are embedded in the membrane and form pores). The second gene of the fragment, sok, does not have a polypeptide product. It encodes labile antisense RNAs that inhibit hok gene translation. The mechanism of the process of directed blocking of the hok gene is described in [Thisted T, Sorensen NS, Gerdes K (1995). "Mechanism of post-segregational killing: secondary structure analysis of the entire Hok mRNA from plasmid R1 suggests a fold-back structure that prevents translation and antisense RNA binding." J. Mol. Biol. 247 (5): 859-73. PMID 7536849].

When E. coli cells divide and the daughter cell loses all copies of the Hok / Sok locus-bearing plasmid, the long-lived protein toxin Hok and its RNA, as well as short-lived Sok antitoxic RNAs, which quickly degrade, then die, and the cell dies. Some E. coli strains may be more resistant to the toxic effects of Hok than others, due to the presence of loci homologous to Hok / Sok in their genome (PMID: 10361310). The probability of death of cells that have lost the plasmid also depends on the initial copy number of the plasmid with the Hok / Sok locus and may depend on the position and orientation of the Hok / Sok locus relative to other transcriptionally active plasmid sites.

A number of studies have described the use of the Hok / Sok locus to increase the segregation stability of plasmids encoding heterologous proteins, including under the strong inducible promoter (US patent 6413768), however, the optimal copy number of such a stabilized plasmid and the optimal position of the Hok / Sok locus relative to other parts of the vector the plasmid obtained in the course of this work remained unknown.

An important step in the preparation of recombinant proteins is the method of purification. Affinity chromatography is one of the most selective methods for protein purification. One step of affinity purification allows to reduce the level of impurities in the solution of the target protein in thousands of times. The standard method for the affinity purification of proteins is the use of sorbents with covalently immobilized antibodies to the protein to be purified or special antibody epitopes artificially added to the sequence of the target protein - tags. Examples of such epitopes to which commercially available monoclonal antibodies have been prepared are FLAG (Einhauer, A. and Jungbauer, A. (2001) The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J. Biochem. Biophys. Methods 49, 455-465) and c-myc (Evan GI, Lewis GK, Ramsay G, Bishop JM. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec; 5 (12): 3610-6.).

Affinity chromatography using immobilized antibodies or other immobilized proteins capable of forming strong complexes with ligand proteins has a number of significant limitations that impede its use in the industrial production of recombinant proteins in microbiological expression systems. The main limitation is the high cost of the antibodies themselves or other pure proteins and the low stability of the sorbents obtained on their basis. Significant limitations include: the inability to regenerate and disinfect sorbents with sodium hydroxide solution, the inability to purify denatured and reduced target proteins, the leak of an immunogenic ligand, the need to elute target proteins with acidic or alkaline solutions.

Some of the drawbacks of conventional affinity chromatography options can be overcome by derivatizing the surface of the chromatographic sorbent with small chemical molecules - protein ligands, which, in turn, are part of the fusion protein with the target protein and are separated from the target protein after affinity purification by treatment with site-specific proteinases. In many cases, such sorbents are inexpensive to manufacture and can withstand treatment with sodium hydroxide solution. Typical examples of systems are maltose binding protein (MBP), glutathione S-transferase (GST), calmodulin binding protein (CBP), strep II (STR). A direct comparison of the applicability of these systems for purification of fusion proteins is given in (Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S. Comparison of affinity tags for protein purification. Protein Expr Purif. 2005 May; 41 (1): 98-105). Affinity purification systems from this group have two significant and irreparable disadvantages - the inevitable contamination of the target protein with an unsplit fusion protein containing immunogenic partner proteins and a decrease in the expression system productivity due to the need for biosynthesis of large amounts of the partner protein.

The only common affinity chromatography method suitable for separating denatured proteins containing only a short tag is metal chelate chromatography. (metal affinity chromatography, Immobilized Metal Ion Affinity Chromatography, IMAC), based on the formation of coordination stable complexes between a transition metal ion coordinated by a chelating agent grafted to the surface of the sorbent and two or three closely spaced histidine residues in the composition of the protein to be purified. (Porath J, Carlsson J, Olsson I, Belfrage G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975 Dec 18; 258 (5536): 598-9; Gaberc-Porekar V, Menart V. Perspectives of immobilized-metal affinity chromatography. J Biochem Biophys Methods. 2001 Oct 30; 49 (1-3): 335-60). When conducting metal chelate chromatography under denaturing conditions, the convergence in space of histidine residues (or other amino acids - electron donors) located in different places of the polypeptide chain becomes difficult and only proteins with artificially created oligohistidine clusters in the composition of the tags exhibit high affinity for the sorbent .

The standard ligand for metal chelate chromatography is the 6xHis peptide (hexahistidine cluster) described in US Pat. No. 4,569,794. The most common type of sorbent for metal chelate chromatography is iminodiacetic acid (IDA) chemical groups immobilized on the surface of dextran spherical granules, other commercially available sorbents use nitrilotriacetic acid (NTA) or carboxymethylaspartate groups.

Since overexpression of heterologous proteins in E. coli cells in most cases leads to the accumulation of the target protein in the bacterial cytoplasm in an insoluble form and the presence of at least one additional N-terminal amino acid residue in the target protein, a unified set of methods for biosynthesis, isolation, primary purification and enzymatic processing of recombinant proteins of medical use, minimally dependent on the nature of the target protein (the so-called technological platform) is more shoy practical interest.

The preferred embodiment of such a technological platform is the use of a short N-terminal tag for affinity purification of the target protein under denaturing conditions and the subsequent separation of such a tag from the target protein using recombinant human site-specific proteinase, i.e., a proteinase with minimal immunogenicity. An example of such a proteinase is the digestive tract enzyme enterokinase, or rather its light chain, which cleaves the polypeptide chain after the Asp-Asp-Asp-Asp-Lys- recognition site and before any amino acid residue except the proline residue. At the same time, the location of the cluster of negatively charged amino acid residues of the enterokinase recognition site near the oligo-histidine cluster leads to a weakening of the interaction of the oligo-histidine cluster with the metal chelate sorbent.

A number of variants of oligohistidine clusters are described in the literature, providing a more tight binding of the purified proteins to the surface of the metal chelate sorbent than the originally described 6xHis cluster. In particular, clusters comprising 6-14 histidine residues interspersed with charged or hydrophobic amino acid residues and described in US patent applications 20100286070 and 20060030007 have relatively high affinity. A significant drawback of these clusters, and especially their most extended variants, is the appearance of plasmid coding for these clusters DNA direct repeats that reduce plasmid stability. Another well-known alternative to the 6xHis cluster is the MAT cluster with the HNHRHKH sequence, including positively charged amino acids and described in US patent application 20060257927, however, its affinity for metal chelating sorbents when negatively charged amino acids are located near the cluster and metal chelate chromatography is carried out under denaturing conditions.

DETAILED DESCRIPTION OF THE INVENTION

The technical problem solved by the authors of the present invention was the development of a highly efficient expression vector that provides increased copying of the vector compared to the traditionally used vector with simultaneous increased segregation resistance with prolonged induction, which allows to increase the level of production of the target protein by conducting productive induction of the target gene for a longer time and start induction at a higher density of culture than in conventional systems.

Another technical problem solved by the authors of the present invention was the use of the above vector for the expression of recombinant proteins fused in a frame with a short N-terminal peptide, followed by ensuring high efficiency of fusion protein purification by metal chelate chromatography and subsequent controlled separation of all amino acids of the indicated peptide specific proteinase.

Another technical problem to be solved within the framework of the present invention was to determine the minimum length of an oligohistidine cluster located in a polypeptide chain near a cluster of negatively charged amino acids and having high affinity for a metal chelate sorbent based on chelating iminodiacetic acid groups during chromatography under denaturing conditions.

The technical result was achieved by creating a plasmid vector based on the pET28a plasmid containing a deletion of the region of the copying restriction element (ROP), replacing the region encoding the additional N-terminal peptide with a region encoding a more effective 10E peptide containing 10 histidine residues and an enterokinase recognition site, and also containing an element of segregation stability Hok / Sok located along the transcription of the target gene after the terminator of transcription of the target gene and oriented in such a way that it is directed e sok gene transcription coincides with the direction of transcription of the target gene.

The vector contains the following elements listed in order:

pBR322 ori — plasmid replication initiation site;

lacI - lactose operon repressor gene;

promoter;

N-leader is a region encoding an N-terminal leader peptide containing a decagystidine cluster and, optionally, a proteinase recognition site;

cloning site (polylinker);

transcription termination site;

a fragment encoding a non-genomic toxin-antitoxin pair - a genetic element that provides segregation stability; and

gene encoding a selection marker.

Elements of the vector are listed in the order of their arrangement. The arrangement of functional elements is essential for the effective operation of the vector.

The promoter can be any strong constitutive or inducible promoter that functions in a bacterial cell, for example, but not limited to the promoter of the bacteriophage T7 RNA polymerase, P _L , P _tac . A bacteriophage T7 RNA polymerase promoter is preferred.

An N-leader is a region encoding an N-terminal leader peptide containing a decagystidine cluster and, optionally, a proteinase recognition site.

The presence of a decahistidine cluster allows metallo-chelate chromatography of the precursor of the target recombinant protein.

The presence of a proteinase site in the N-terminal leader peptide of the target recombinant protein precursor is necessary. This site allows the separation of the N-terminal leader peptide. However, the presence of a fragment encoding a proteinase recognition site in the vector described above is not necessary. Said fragment encoding a proteinase recognition site can be incorporated into a construct for expressing a target protein precursor together with an open reading frame (OPC) of the target protein. For this, adapter primers are used, one of which contains a suitable restriction site for cloning, a fragment encoding a proteinase recognition site located in the same reading frame next to the first amino acid of the target protein, or directly in front of it, and a portion identical to the 5′-region of the OPC target squirrel; and the second primer contains a suitable restriction site for cloning, and a plot complementary to the 3′-region of the OPC of the target protein. A DNA fragment containing a fragment encoding a proteinase recognition site and an OPC of a target protein can be obtained by PCR using the adapter primers described above.

The design of such adapter primers for a particular target gene is a routine procedure and one skilled in the art can easily design and synthesize such primers.

In the case where the fragment encoding the proteinase recognition site is in the vector described above (in the N-leader region), the OPCs of the target protein are ligated into the indicated vector so that the N-leader and OPC are in the same reading frame. Gene expression of the target protein in the resulting plasmid leads to the formation of a precursor, cleavage of which by the proteinase leads to the formation of the target protein containing several additional amino acids at the N-terminus.

In the case where the fragment encoding the proteinase recognition site located immediately before the first amino acid of the target protein is ligated in the fragment containing the OPC of the target protein and obtained using adapter primers as described above, expression of the target protein gene in the resulting plasmid leads to the formation of a precursor, cleavage of which by proteinase leads to the formation of the target protein that does not contain any additional amino acids at the N-terminus. This approach allows you to get native target proteins that can be used for therapeutic purposes.

As a specific proteinase used for cleavage of the leader peptide from the obtained hybrid protein after its purification by metal chelate chromatography, human enterokinase, cattle enterokinase, human specific thrombin proteinases, factor XA, specific protein engraving of the TEV tobacco engraving virus, specific rhinovirus proteinase can be used. human HRV3C (for the production of therapeutic proteins, it is preferable to use specific human proteinases, since the theologous proteins in the therapeutic protein will be immunogenic). However, specific proteinases are not limited to these proteinases.

The transcription termination site may include the T7 bacteriophage transcription termination site (T7term), E. coli rrnC, rrnBt1 and rrnBt2 transcription termination sites, the sequence of the vesicular stomatitis virus VSV, the human preproparathyroid hormone (PTH) gene, but not limited to them. The T7 bacteriophage transcription termination site (T7term) is preferred.

Examples of non-genomic toxin-antitoxin pairs are the hok / sok system stabilizing the R1 plasmid in E. coli, the FlmA / FlmB system, the hok / sok homolog stabilizing the plasmid F in E. coli, and the like. A hok / sok system is preferred.

As a selection marker, an antibiotic resistance gene can be used, for example, but not limited to kanamycin, ampicillin, tetracycline, chloramphenicol. The kanamycin resistance gene is preferred.

A specific embodiment of the vector according to the present invention is a pHYP vector in which the promoter is a T7 bacteriophage RNA polymerase promoter, the transcription termination site is the T7 bacteriophage transcription termination site, the specific proteinase recognition site is an enterokinase recognition site, and the gene encoding the selection marker is kanamycin resistance gene.

The vector consists of 5151 po and contains unique (i.e. the only) recognition sites for restriction endonucleases: XhoI (529), HindIII (544), NheI (602).

The functional diagram of the vector indicating the coordinates of the functional elements is shown in Fig. 9, and the complete nucleotide sequence of this vector is presented in the Sequence Listing under the number SEQ ID NO: 40. The vector was obtained using standard genetic engineering methods and chemically synthesized oligonucleotides [Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. 2nd ed. New York, NY: Cold Spring Harbor Laboratory Press; 1989].

Using the created vector, it is possible to transform a bacterial cell, preferably a bacterium belonging to the genus Escherichia, susceptible to such a transformation by the specified vector. 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.

"Transformation of a cell by a vector" means the introduction of a vector 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 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, “bacterial cell producing a recombinant protein precursor” means a bacterial cell having the ability to produce and accumulate a recombinant protein precursor containing a decagystidine cluster and an enterokinase recognition site when the bacterial cell of the present invention is grown in the specified nutrient medium. The specified recombinant protein precursor can accumulate in the specified cell, preferably in the form of inclusion bodies.

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 of the present invention is Escherichia coli strain BL21 [DE3], but the range of suitable strains is not limited thereto.

The method according to the present invention includes cultivating in a suitable nutrient medium a bacterium containing the vector described above containing the target gene encoding the target recombinant protein fused to the N-terminal peptide containing the decagystidine cluster and a human proteinase recognition site; purification of fusion protein by metal chelate chromatography; and separating the peptide from the target recombinant protein using a site-specific human proteinase.

Features of the obtained vector, expression plasmids based on it, intermediate plasmids and the results of their practical application are shown in the following Figures.

Short Description of Shapes

The Figure 1 shows the scheme for obtaining plasmids p6E, p8E, pME, p10E, p10E-HU, p10E-HD, p10E-HDR, p10Erop ^- , p10E-HUrop ^- , pHYP, p10E-HDRrop ^- . Designations: the dashed line indicates the PCR stages, the solid line indicates the restriction-ligation stages, the restriction endonucleases used for cloning are indicated as plasmids, HS-PCR is the PCR product encoding the hok / sok (host killing / suppressor of killing) region of the natural plasmid R1 Escherichia coli.

Figure 2 shows a map of the expression plasmid p10E. 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; "N-leader" is an open reading frame (OPC) of the N-terminal leader containing a decahystidine cluster. 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.

Figure 3 shows a map of the expression plasmid p10E-HU. 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; "N-leader" is an open reading frame (OPC) of the N-terminal leader containing a decahystidine cluster. Hok / Sok - plot hok / sok (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli; Hok is a product of the hok gene (toxin), Sok is a product of the sok gene. 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.

Figure 4 shows a map of the expression plasmid p10E-HD. The following notation is used: "pBR322ori" region of the beginning of replication of plasmid pBR322; "ROP" - gene of the RNA-organizing protein Rop; "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; “N-leader” is an open reading frame (OPC) of an N-terminal leader containing a decahystidine cluster. Hok / Sok - plot hok / sok (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli; Hok is a product of the hok gene (toxin), Sok is a product of the sok gene. 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.

Figure 5 shows a map of the expression plasmid p10E-HDR. The following notation is used: "pBR322ori" region of the beginning of replication of plasmid pBR322; "ROP" - gene of the RNA-organizing protein Rop; "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 a sequence encoding a repressor of a lactose operon; “N-leader” is an open reading frame (OPC) of an N-terminal leader containing a decahystidine cluster. Hok / Sok - plot hok / sok (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli; Hok is a product of the hok gene (toxin), Sok is a product of the sok gene. 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.

Figure 6 shows a map of the expression plasmid p10Erop-. The following notation is used: "pBR322ori" region of the beginning of replication of plasmid pBR322; "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 a sequence encoding a repressor of a lactose operon; “N-leader” is an open reading frame (OPC) of an N-terminal leader containing a decahystidine cluster. Hok / Sok - plot hok / sok (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli; Hok is a product of the hok gene (toxin), Sok is a product of the sok gene. 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.

Figure 7 shows a map of the expression plasmid p10E-HUrop-. The following notation is used: "pBR322ori" region of the beginning of replication of plasmid pBR322; "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; "N-leader" is an open reading frame (OPC) of the N-terminal leader containing a decahystidine cluster. Hok / Sok - plot hok / sok (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli; Hok is a product of the hok gene (toxin), Sok is a product of the sok gene. 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.

Figure 8 shows a map of the expression plasmid p10E-HDrop-. The following notation is used: "pBR322ori" region of the beginning of replication of plasmid pBR322; "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; "N-leader" is an open reading frame (OPC) of the N-terminal leader containing a decahystidine cluster. Hok / Sok - plot hok / sok (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli; Hok is a product of the hok gene (toxin), Sok is a product of the sok gene. 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.

Figure 9 shows a map of the expression plasmid pHYP. The following notation is used: "pBR322ori" region of the beginning of replication of plasmid pBR322; "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; "N-leader" is an open reading frame (OPC) of the N-terminal leader containing a decahystidine cluster. Hok / Sok - plot hok / sok (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli; Hok is a product of the hok gene (toxin), Sok is a product of the sok gene. 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.

The Figure 10 presents the results of measuring the proportion of cells containing ROP ⁺ plasmids after 8 and 24 hours of induction of 2 mm IPTG. The number of independent experiments is 7-12; confidence intervals for p = 0.05 are given.

The Figure 11 presents the results of measuring the proportion of cells containing ROP ⁺ plasmids without insertion after 24 hours of induction of 0.2 mm IPTG. The number of independent experiments is 3-4; confidence intervals are given for p = 0.05.

Figure 12 shows the results of measuring the proportion of cells containing ROP ^- a plasmid without an insert, after 24 hours of induction with IPTG 0.2 mM. The number of independent experiments is 3-4; confidence intervals are given for p = 0.05.

The essence and industrial applicability of the present invention are illustrated by the following examples.

Example 1. Obtaining plasmids pAL-HS

The hok / sok site (host killing / suppressor of killing) of the natural plasmid R1 Escherichia coli, the sequence of which was deposited in the GeneBank public database (GenBank X05813), was obtained by the polymerase chain reaction using AS-HOS-F5 synthetic oligonucleotides (SEQ ID NO: one); AS-HOS-F4 (SEQ ID NO: 2); AS-HOS-F3 (SEQ ID NO: 3); AS-HOS-F2 (SEQ ID NO: 4); AS-HOS-F1 (SEQ ID NO: 5); AS-HOS-R1 (SEQ ID NO: 6); AS-HOS-R2 (SEQ ID NO: 7); AS-HOS-R3 (SEQ ID NO: 8); AS-HOS-R4 (SEQ ID NO: 9); AS-HOS-R5 (SEQ ID NO: 10) on a Tertsik MS2 instrument (DNA Technology, Russia). The nucleotide sequences of primers 5'-3 ':

The hok / sok region of the plasmid R1 E coli (GenBank X05813) is presented in the Sequence Listing under the number SEQ ID NO: 11.

The hok gene encodes a 52 amino acid polypeptide toxin (SEQ ID NO: 12) that causes membrane potential dissipation and has stable mRNA, while transcription of the sok antitoxin gene leads to the appearance of short-lived short antisense RNAs that bind to the hok gene mRNA and inhibit its translation. When a bacterial cell loses a plasmid containing an Hok / Sok locus, transcription of the sok element ceases and its mRNA decays rapidly and translation of the long-lived Hok gene is restored, resulting in the death of the bacterial cell.

The sequence of the PCR product containing the hok / sok region of E. coli plasmid R1 is shown in the sequence list under the number SEQ ID NO: 13. External primers AS-HOS-F5 (SEQ ID NO: 1) and AS-HOS-R5 (SEQ ID NO: 10) contain recognition sites for restriction endonucleases PspCI, BamHI, SphI.

The polymerase chain product was purified from a 1% agarose gel using the Wizard SV Gel and PCR Clean-Up System "kit (Promega, USA) according to the manufacturer's protocol. The purified products were ligated into the PAL-TA plasmid (ZAO Eurogen, Russia) with T4 DNA ligase (Fermentas , Lithuania) Ligation was carried out in a volume of 10 or 20 μl, with a molar ratio of the vector and insert of 1:10, for 2-20 hours at room temperature.The obtained ligase mixtures transformed E. coli cells of strain DH5α and plated on solid agar medium containing ampicillin at a concentration of 100 μg / 1 ml of agar, and placed in a thermostat at 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 M13rev (SEQ ID NO: 14) and M13dir (SEQ ID NO: 15) The selected clones were grown in 5 ml of 2xYT-Amp nutrient broth and plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit (Fermentas, Lithuania) according to the manufacturer's protocol. For the resulting PAL-HS constructs, the nucleotide sequence was determined by PCR sequencing using primers for the sequence of the vector SP6 (SEQ ID NO: 16) and T7prom (SEQ ID NO: 17).

Example 2. Obtaining expression plasmids p6E, p8E, pME, p10E and comparison of the properties of leader peptides in metal chelate chromatography under denaturing conditions

The sequences encoding fragments of the N-terminal additional peptides (tags) 6E (SEQ ID NO: 18), 8E (SEQ ID NO: 19), 10E (SEQ ID NO: 20), MAT (SEQ ID NO: 21) were obtained by the method annealing of two partially complementary oligonucleotides with the formation of protruding “sticky” 5′-ends. For this, the corresponding pairs of oligonucleotides were introduced into the test tube, 100 pmol each in a total volume of 10 μl, heated to 95 ° C and slowly cooled to room temperature.

To obtain tag 6E, primers AD-6H-NcoF (SEQ ID NO: 22) and AD-6H-NheR (SEQ ID NO: 23) were used; primers AD-8H-NcoF (SEQ ID NO: 24) and AD-8H-NheR (SEQ ID NO: 25) were used to obtain tag 8E; primers AD-10H-NcoF (SEQ ID NO: 26) and AD-10H-NheR (SEQ ID NO: 27) were used to obtain tag 10E; primers AD-MAT-NcoF (SEQ ID NO: 28) and AD-MAT-NheR (SEQ ID NO: 29) were used to obtain the MAT tag.

The recipient plasmid pET28a (+) (Novagen, USA) was sequentially digested with NcoI and NheI endonucleases. For this, two 0.5 μg aliquots of the purified plasmid were incubated for 2 hours in a water thermostat at 37 ° C with one of the two restriction endonucleases, the completeness of linearization of the plasmids was controlled by agarose gel electrophoresis, after which a second restriction enzyme was added and incubated for another 2 hours. Then the samples were combined, restriction enzymes were inactivated by heating at 65 ° C for 20 minutes. The restriction enzyme digested plasmid was treated with an equal volume of a 1: 1 phenol-chloroform mixture and the aqueous phase was reprecipitated with three volumes of chilled ethanol. It was centrifuged for 10 minutes at a speed of 13,200 rpm at room temperature, the precipitate was washed with a 70% ethanol solution, dissolved in water and used to set up a dosing reaction at a working concentration of 10 ng / μl, inserts — corresponding oligonucleotide duplexes were used in a molar ratio of vector: insert 1 :10. The reaction was carried out using T4 DNA ligase (Fermentas, Lithuania) according to the manufacturer's procedure. The ligase was inactivated by heating the reaction mixture for 20 min at + 65 ° C and transformation of competent E. coli cells of strain DH5α was carried out according to the procedure described in [Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. 2nd ed. New York, NY: Cold Spring Harbor Laboratory Press; 1989]. Colonies of E. coli transformants were grown on agar medium containing 30 μg / ml kanamycin and the colonies were analyzed by clone PCR using T7t oligonucleotides (SEQ ID NO: 30) and T7prom (SEQ ID NO: 17). Selected colonies containing an insert of the expected size were used to inoculate liquid cultures with 5 ml of 2xYT-kanamycin nutrient broth, plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit (Fermentas, Lithuania) according to the manufacturer's protocol, and the nucleotide sequence for the insertion region was determined by PCR sequencing with using T7t primer (SEQ ID NO: 30).

The sequences of the obtained vectors p6E, p8E, p10E, pME are presented in the List of sequences under the numbers SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, respectively.

To test the properties of the N-terminal peptides encoded by vectors, expression plasmids were obtained that encoded a synthetic gene for the deletion variant of human tissue plasminogen activator containing 355 of 527 amino acids of natural human TAP (amino acids 1-3 and 176-527) and an enterokinase cleavage site (SEQ ID NO : 43) fused in frame with the corresponding peptides.

The expression plasmid p10E-tPA was obtained as described in the Reference example below, plasmids p6E-tPA, p8E-tPA and pME-tPA were obtained similarly to p10E-tPA. For all four plasmids obtained, the insertion region was sequenced; in further work, clones that did not contain point mutations were used.

Fusion protein producing strains were obtained by transforming expression plasmids into cells of strain BL21 [DE3] (Novagen, USA) by electroporation. Conducted analytical expression of the target genes. In all four cases, the majority of the target proteins accumulated in an insoluble form in the inclusion bodies, gene expression levels did not differ significantly. Inclusion Taurus preparations for four target fusion proteins 6E-tPA, ME-tPA, 8E-tPA, 10E-tPA produced by strains BL21 [DE3] / p6E-tPA, BL21 [DE3] / pME-tPA, BL21 [DE3] / p8E -tPA, BL21 [DE3] / p10E-tPA, respectively, was prepared as follows. Biomass pellets were separated by centrifugation, cell pellets were resuspended in 20 ml of solution A (50 mM Tris pH 7.4 2 mM EDTA), lysozyme up to 10 μg / 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. Precipitation was separated by centrifugation for 10 minutes at 18,000 rpm. Precipitation was resuspended in 20 ml of solution A, NP-40 detergent was added to 1%, the suspensions were treated with an ultrasonic disperser until the sediment particles larger than 1 mm disappeared, inclusion bodies were separated by centrifugation for 10 min at 20,000 rpm. The resulting precipitates of inclusion bodies were 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 preparations of enriched inclusion bodies were stored at -70 ° C.

Samples of 50 mg Taurus were each solubilized for inclusion of each protein variant in a solution of 6M guanidine hydrochloride, 50 mM Tris-HCl, 50 mM β-mercaptoethanol, pH = 8.0. A 10% suspension of inclusion bodies was incubated for 30 min at + 37 ° C with stirring and the cell debris sediment was separated by centrifugation. A column for metal chelate chromatography 1 ml of HisTrap (GE Healthcare, USA) containing an agarose sorbent with grafted iminodiacetic acid groups (IDA) and immobilized nickel ions was prepared for work. The column was equilibrated with solution A containing 6 M guanidine hydrochloride, 50 mM Tris-HCl, 5 mM β-mercaptoethanol, pH = 7.5, and solutions of the corresponding fusion proteins were applied at a flow rate of 0.2 ml / min. The fraction of proteins not bound to the column was collected and the column was washed with solution A until the detector baseline (about 10 column volumes) stabilized at a flow rate of 1 ml / min. Stepwise protein elution was carried out in portions of 5 ml of solutions containing 50, 100, 200, 500 mM imidazole in solution A, pH = 7.5. The eluates were collected and the immobilized nickel ions (and the protein molecules coordinated by them) were removed from the column of 5 ml of solution A, additionally containing 50 mM EDTA-Na, the eluate was collected.

Proteins were precipitated from solutions of guanidine hydrochloride to conduct SDS-PAGE analysis of proteins; for this, trichloroacetic acid up to 7% was added to equal aliquots of all eluates and an equivalent aliquot of the slip fraction, solutions were mixed, incubated for 30 min at room temperature, protein precipitates were separated by centrifugation, the precipitates were washed twice with chilled acetone and the precipitates were dried at room temperature.

SDS-PAGE analysis of protein fractions was performed, the gel was stained with a Kumasi blue colloidal solution (Fermentas, Lithuania), the excess dye was washed with purified water, and the protein content in the gel plate was determined by densitometry using a flatbed scanner in transmission mode. The obtained digital image of the gel with a color depth of 16 bits / channel was analyzed using the TotalLab program (Nonlinear Dynamics, United Kingdom) and the peak volumes corresponding to the bands of the target protein in the fractions were determined. The distribution of the target protein in fractions for the studied tags are shown in table 1.

Table 1 The proportion of target proteins in the elution fractions during metal chelate chromatography under denaturing conditions. Expression Product 6E-tPA ME-tPA 8E-tPA 10E-tPA Elution with 50 mM imidazole 37.3% 16.0% 2.3% 1.2% Elution with 100 mM imidazole 20.8% 22.7% 1,0% 3.0% Elution 200 mM imidazole 27.7% 29.1% 19.5% 15.6% Elution 500 mM imidazole 7.3% 27.2% 71.1% 72.5% Elution EDTA 6.9% 5.0% 6.1% 7.7%

It was found that the 10EtPA protein has the highest resistance to elution with increasing concentrations of imidazole, while the fraction of the target protein not eluted with 500 mM imidazole and eluted upon removal of immobilized nickel ions using EDTA is practically independent of the length and type of the oligohistidine cluster and remains insignificant. Since most of the E. coli proteins visible on the gel, which bind to the metal chelate sorbent under denaturing conditions, elute at an imidazole concentration of up to 200 mM inclusive, the preferred option for purification of the target proteins is the elution of impurity proteins at an imidazole concentration of 200 mM and subsequent elution of the target protein at an imidazole concentration 500 mM. In this mode of chromatography, the best yield is recorded for proteins with a 10E tag.

To verify the obtained data, a producer strain encoding tPA without a tag, but with an additional methionine residue at the N-terminus of the protein, was obtained. When conducting metal chelate chromatography under denaturing conditions for this protein, it was found that the target protein does not bind to the metal chelate sorbent and is completely found in the fraction of unbound proteins. Thus, the intrinsic properties of the denatured tPA polypeptide with reduced disulfide bonds do not significantly affect the binding of fusion proteins to a metal chelate column.

Presumably, when using tags that do not contain a cluster of acidic amino acids (DDDDK enterokinase recognition site) near an oligo-histidine cluster, the elution profile of polypeptides with oligo-histidine clusters of different lengths will differ from the above and the resistance of fusion proteins to elution can increase up to the inability to elute the target protein with a solution with 500 mM imidazole.

Example 3. Obtaining vector plasmids p10E-HU, p10E-HD, p10E-HDR and comparison of their segregation stability

To obtain the p10E-HU vector, the recipient plasmid p10E was digested with BglII and SphI endonucleases. The donor plasmid PAL-HS was digested with BamHI and SphI endonucleases. Restriction products were isolated from 1% agarose gel using the WizardSV Gel and PCR Cean-Up System kit (Promega, USA), after which T4 DNA was ligated with ligase and E. coli cells of strain DH5a were transformed. E. coli colonies were analyzed by clone PCR using AS-HOS-F5 oligonucleotides (SEQ ID NO: 1) and AS-HOS-R5 (SEQ ID NO: 10). Selected clones were grown in 5 ml of 2xYT-Kan, plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit. The p10EHU sequence was determined by PCR sequencing using a T7t primer (SEQ ID NO: 30).

To obtain the vectors p10E-HD and p10E-HDR, the recipient plasmid p10E was digested with the endonuclease PsiI. The donor plasmid PAL-HS was digested with PspCI endonuclease (Fermentas, Lithuania). Restriction products were isolated from 1% agarose gel, and E. coli cells of strain DH5α were donated and transformed as described above. E. coli colonies were analyzed by clone PCR using AS-HOS-F5 oligonucleotides (SEQ ID NO: 1) and AS-HOS-R5 (SEQ ID NO: 10). PCR from primers T7prom (SEQ ID NO: 17) and AS-HOS-F5 (SEQ ID NO: 1) or AS-HOS-R5 (SEQ ID NO: 10) was used to determine the orientation of the insert during the initial screening of clones. Plasmid p10E-HD contained the insert in the direct orientation, plasmid p10E-HDR in the reverse orientation. Selected clones were grown in 5 ml of 2xYT-Kan nutrient broth, plasmid DNA was isolated using the Gene JET Plasmid Miniprep Kit. The sequence of p10EHD and p10EHDR was determined by PCR sequencing using a T7prom primer (SEQ ID NO: 17).

The sequences of the obtained plasmids p10E-HU, p10E-HD, p10E-HDR are presented in the sequence Listing under the numbers SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, respectively. Plasmids differ in position and orientation of the Hok / Sok segregation stabilization locus. Since the locus is close to the active T7, lac, and kanR promoters, it was suggested that transcription of plasmid DNA sites that capture the region of the Hok / Sok locus initiated by these promoters could significantly affect the relative level of transcription of the hok and sokSok genes and, thus, change the properties locus.

To compare the segregation stability of the obtained plasmids, an insertion of a deletion variant of tissue plasminogen activator with an additional N-terminal tag of 10E (tPA) was cloned into them. Cloning of the tPA minigene into the created vectors was performed as described in Example 2. The tPA producing strains in the corresponding vectors and strains carrying the studied plasmids without the protein coding insert were obtained by electroporation of E. coli BL21 [DE3] strain.

We conducted the cultivation of the studied strains according to the following general scheme:

1. A strain from a frozen museum was plated with a draining stroke on a Petri dish with 2xYT agar medium containing 30 μg / L kanamycin and 1% glucose. Cultivated for 14-18 hours

2. Single colonies of the phenotype typical for BL21 [DE3] were selected and transferred to test tubes with 5 ml of 2xYT liquid medium containing 30 μg / l kanamycin and 1% glucose. The culture was grown for 16 hours. 1 ml of culture was transferred to a sterile microtube and the medium with antibiotic residues was separated by centrifugation. Resuspend cell pellet in 1 ml of 2xYT medium.

3. Inoculable 50 μl of the obtained suspension of bacterial cells in 5 ml of 2xYT medium containing 0.1% glucose but not containing kanamycin, increased for 2 hours, after which IPTG was added up to 2 mm and cultivation continued for 24 hours.

4. 8 hours after the start of induction, an aliquot of the culture was taken, dilutions of 10 thousand and 1 million were made using sterile 2xYT medium. 100 μl of the obtained dilutions were plated on plates with 2xYT medium with and without 30 mg / l kanamycin. Cultures were grown in for 14-18 hours and the grown colonies were counted.

5. Aliquots of the liquid culture were selected from step 4 24 hours after induction, dilutions according to claim 4 were prepared and plated, and colonies were counted.

The results of measuring the proportion of cells that retained plasmids 8 and 24 hours after the start of induction are shown in FIG. 10. For all variants of the insertion of the Hok / Sok locus, there was no statistically significant change in the segregation stability of the plasmid. The segregation resistance measurement was repeated for strains transformed with plasmids without a coding insert, and a culture without antibiotic was used in the presence of 0.2 mM IPTG for 24 hours. The results of measuring the fractions of cells that retained the plasmid are shown in FIG. 11. The segregation stability of plasmids that did not contain an insert also did not have statistically significant differences and did not have significant differences from the stability of plasmids encoding a non-toxic protein. It was suggested that the level of expression of the hok gene is insufficient to induce cell death during plasmid loss, or that the expression level of the sok antitoxin gene is too high, or the functioning of the hok gene is hindered by the expression of genes homologous to sok located in the BL21 cell genome [DE3]. The most obvious way to increase the level of expression of the hok and sok genes present in constructed plasmids is to increase the copy number of plasmids in cells, realized by removing the genetic control element of copy number ROP.

Example 4. Obtaining plasmids p10Erop ^- , p10E-HUrop ^- , p10E-HDRrop ^- , pHYP and comparison of their segregation stability

The removal of the DNA fragment encoding the ROP element was performed by restriction of plasmids p10E (SEQ ID NO: 33), p10E-HU (SEQ ID NO: 35), p10E-HD (SEQ ID NO: 36), p10E-HDR (SEQ ID NO: 37) two restriction endonucleases that cleave DNA to form blunt ends - FspAI (Fermentas Lithuania) and Bst1107I (Fermentas Lithuania). Fragments after cleavage were isolated from agarose gel using the Wizard SV Gel and PCR Clean-Up System kit (Promega, USA), after which T4 DNA was ligated with a ligase (Fermentas, Lithuania) according to the manufacturer's method. Ligation products were digested with the same endonucleases, after which E. coli strain DH5α was transformed. Clones of transformants were grown in 5 ml of 2xYT-Kan nutrient broth and plasmid DNA was isolated using a GeneJET Plasmid Miniprep Kit reagent kit (Fermentas, Lithuania). The identity of the clones was confirmed by restriction analysis using the same FspAI and Bst1107I endonucleases for lack of fragment cleavage.

The sequences of the obtained plasmids p10Erop ^- , p10E-HUrop ^- , pHYP, p10E-HDRrop ^{- are} presented in the List of sequences under the numbers SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively.

An analysis of the segregation stability of the obtained plasmids was carried out as described in Example 3. The measurement results are shown in Figure 12. It was found that the plasmid pHYP (Hok-Sok element in the HD position) provides an approximately twofold and statistically significant (p <0.05) increase in the proportion cells carrying the plasmid, compared with all other studied plasmids, with prolonged induction of strains using IPTG. Thus, an expression vector was created that provides increased segregation stability during cultivation in the absence of an antibiotic and in the presence of the target gene expression inducer, and also allows purification of target proteins by metal chelate chromatography under denaturing conditions with an increased yield of the product.

Reference example. Obtaining plasmid p10E-tPA

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: 44) and AD-10H-NheR (SEQ ID NO: 45) 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 subsidized 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: 44) and T7t (SEQ ID NO: 46). Selected clones were grown in 5 ml of 2xYT-Kan nutrient broth, plasmid DNA was isolated using the Gene JET 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: 46).

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, then 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 preliminarily digested with restriction enzymes NheI and HindIII of the fragment SEQ ID NO: 43 corresponding to the tPA minigen and containing the enterokinase cleavage site and the recipient plasmid was carried out as described above. After that, cells of E. coli strain DH5α were transformed, as described above. The fragment of SEQ ID NO: 43, corresponding to the tPA minigen and containing the enterokinase cleavage site, was obtained by PCR using a set of overlapping primers. Alternatively, this fragment can be obtained using the cloning technology of Sloning BioTechnology, described in PCT application WO2005071077.

E. coli colonies were analyzed by PCR from clones from primers T7prom (SEQ ID NO: 47) and T7t (SEQ ID NO: 46). 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: 47) and T7t (SEQ ID NO: 46). 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.

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 (9)

1. A vector for producing a vector for expression in a bacterial cell of a precursor of a target recombinant protein fused to an N-terminal peptide containing a decagystidine cluster and a proteinase recognition site essentially consisting of:
- site of replication initiation pBR322 ori;
- lactose operon repressor gene;
- bacterial promoter;
a region encoding an N-terminal leader peptide containing a decagystidine cluster and, optionally, a proteinase recognition site;
- cloning site (polylinker);
- plot termination of transcription functioning in a bacterial cell;
- a fragment encoding a non-genomic pair of toxin-antitoxin, while the antitoxin gene is oriented in such a way that the direction of its transcription coincides with the direction of transcription of the target gene; and
- a gene encoding a bacterial selection marker. 2. The vector according to claim 1, characterized in that the promoter is a T7 bacteriophage RNA polymerase promoter, the transcription termination site is the T7 bacteriophage transcription termination site, the proteinase recognition site is an enterokinase recognition site, a fragment encoding a non-genomic toxin-antitoxin pair, is the hok / sok system, and the gene encoding the selection marker is the kanamycin resistance gene. 3. The vector according to claim 1, containing the sequence shown in the List of sequences under the number SEQ ID NO: 40. 4. A vector for expression in a bacterial cell of a precursor of a target recombinant protein fused to an N-terminal peptide containing a decagidine cluster and a proteinase recognition site essentially consisting of:
- site of replication initiation pBR322 ori;
- lactose operon repressor gene;
- bacterial promoter;
a region encoding an N-terminal leader peptide containing a decagystidine cluster and, optionally, a proteinase recognition site;
- a cloning site (polylinker) with an open reading frame inserted encoding the indicated target recombinant protein, and if the proteinase recognition site is missing in the indicated vector, the specified site is pre-introduced before the open reading frame;
- a transcription termination site functioning in a bacterial cell;
- a fragment encoding a non-genomic pair of toxin-antitoxin, while the antitoxin gene is oriented in such a way that the direction of its transcription coincides with the direction of transcription of the target gene; and
- a gene encoding a bacterial selection marker. 5. The vector according to claim 4, characterized in that the promoter is a T7 bacteriophage RNA polymerase promoter, the transcription termination site is the T7 bacteriophage transcription termination site, the proteinase recognition site is an enterokinase recognition site, a fragment encoding a non-genomic toxin-antitoxin pair, is the hok / sok system, and the gene encoding the selection marker is the kanamycin resistance gene. 6. A bacterium belonging to the genus Escherichia and containing the vector according to claim 4, is a producer of the precursor of the target recombinant protein containing an N-terminal peptide containing a decagystidine cluster and a proteinase recognition site. 7. The bacterium according to claim 6, characterized in that the proteinase recognition site is an enterokinase recognition site. 8. A method of obtaining a recombinant protein, including:
- culturing the bacteria according to claim 6 in a nutrient medium suitable for the specified bacteria,
- purification of fusion protein by metal chelate chromatography and
- separation of the peptide from the target recombinant protein using site-specific proteinase. 9. The method of claim 8, wherein the site-specific proteinase is enterokinase.

Images