RU2604796C1 - EXPRESSION SYSTEM AND METHOD OF PRODUCING NON-MODIFIED RECOMBINANT PROTEINS IN Escherichia coli USING IT - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology. DNA construct is disclosed, which codes fused precursor protein, in which auxiliary amino acid sequence is bound to N-end of mature target polypeptide sequence with transition site intended for identification and splitting of hybrid precursor by specific protease to form non-modified protein of interest. Above site represents truncated at N-end by 23 amino acid residues SUMO protein of S. cerevisiae (Smt3), which includes site of splitting with SUMO-hydrolase (Ulp1). On basis of DNA construct coding version of precursor, which comprises glutathione-S-transferase (GST) as auxiliary sequence and negatively charged peptide and human interferon (IFN) α2b as target protein recombinant E. coli strain is obtained. Method of producing non-modified IFN α2b using above E. coli strain is also disclosed.

EFFECT: group of inventions enables to obtain target protein with high purity and activity.

7 cl, 9 dwg, 2 tbl, 5 ex

Description

The invention relates to biotechnology, in particular to the production of recombinant polypeptides through an intermediate form of a hybrid protein. A DNA construct encoding a fusion precursor protein is proposed in which the auxiliary amino acid sequence is linked to the N-terminus of the mature target polypeptide sequence with a transition region for recognizing and cleaving the hybrid precursor with a specific protease to form an unmodified mature form of the protein of interest. According to the invention, said region is a SUMOSac protein truncated from the N-terminus into 23 amino acid residues. cerevisiae (Smt3), which contains the cleavage site of SUMO hydrolase (Ulp1). The specific forms of recombinant DNAs and the hybrid proteins encoded by them, including various fusion partners, were obtained, characterized and tested. Based on the DNA construct encoding a variant of the precursor, which includes, as an auxiliary sequence, glutathione S-transferase (GST) and a negatively charged peptide, and the recombinant E. coli strain is obtained as the target protein, human interferon (IFN) α2b, and a method is described obtaining unmodified IFN α2b with its use.

Introduction

Gram-negative bacterium Escherichiacoli is one of the most widely used host microorganisms for the industrial production of recombinant proteins. However, it is well known that the expression of heterologous proteins in E. coli cells is associated with a number of problems that must be solved in the process of their preparation. In particular, if after the synthesis of authentic (intrinsic) proteins, the additional N-terminal methionine residue due to the translation mechanism is effectively removed in most cases by bacterial enzymes, then, when foreign proteins are expressed, partial cleavage occurs in the best case. Additional methionine at the N-terminus of the recombinant protein may not adversely affect its properties, however, it is known that its presence in some cases leads to a decrease in stability and an increase in the immunogenicity of proteins (Ben-Bassat A., Bauer K., 1987, Nature 326, 315) and even to the loss of their biological activity (Rabbani et al., 1988, J Biol Chem, 263: 1307-1313). This, in particular, served as the basis for the recognition of IFN α2 as a genomically modified product and the restriction of the production and use of preparations containing it in European countries (Eur. Pharm., 5 ^th ed .: 2004).

Currently, the most common method for synthesizing heterologous proteins with the “correct” N-terminus in E. coli is to obtain the target protein through a hybrid precursor, from which the final product is subsequently cleaved by treatment with a specific protease. It is important that the application of this methodology, in addition to the main task of removing the additional methionine residue, allows us to solve a number of other problems arising from the expression of heterologous proteins in E. coli, including the prevention or reduction of their proteolytic degradation in bacterial cells, as well as the possibility of “errors” »In the formation of the native structure of protein molecules; if necessary, to overcome the low solubility of the hybrid protein and significantly simplify the purification methodology of the target product.

In view of the foregoing, it seems obvious that the task of developing new genetic constructs and expression systems for obtaining heterologous proteins of interest through E. coli through hybrid precursors remains relevant.

State of the art

The fusion partners most commonly used in hybrid precursors of target proteins are histidine tag (6-10 His), maltose binding protein (MBP) (Kapust R et al., 1999, Protein Sci., 8, 1668-1674), glutathione S-transferase (GST) (Rabhi-Essafi I. et al., 2007, Protein Eng. Des. Sel. 20, 201-209; Smith D., 1988, Gene 67, 31-40; Artika M. et al ., Am. J. Biochem. Biotechnol., 2013, 9 (4), 423-429) a highly charged peptide (EP 0089626), SUMO protein (T. Butt et al., Protein Expr. Purif. 43, 1-9 ; Zhu F. et al., World J. Microbiol. Biotechnol., 2013, 29, 319-325; Peciak K. et al., 2005, Protein Exp. Purif., 43, 1-9; Panavas T. et al ., 2009, Methods Mol Biol., 497, 303-317) and others. Moreover, the general positive result of combining any of the following of auxiliary polypeptides with the target protein is a certain increase in the degree of protection of the latter from proteolytic degradation, while in the rest, each of the partners brings individual features to the hybrid protein.

So, it is known that His-tag provides the ability to separate the hybrid protein using metal (Ni, Cu, Zn, Co) - chelate affinity chromatography; MBP increases solubility and promotes proper protein refolding; (+) or (-) - a charged peptide allows you to directionally change the charge of the protein molecule (pI); GST - to increase the yield of the precursor and its solubility, and also to use the affinity affinity of GST for glutathione-containing sorbents to “capture” the hybrid protein.

As for the use of ubiquitin-like proteins, in particular SUMO protein, as a fusion partner of interest to the polypeptide, the main feature of this type of partners is that, unlike all of the above, the resulting hybrid precursor does not need to introduce a separate protease site for cleavage the target product, since such a site is represented in the SUMO molecule itself. Among the main advantages of SUMO-containing hybrid precursors are the high specificity of hydrolysis, as well as the fundamental possibility of increasing the solubility of the "hybrid". The correct cleavage of the hybrid precursor, including SUMO, has been confirmed by numerous examples, including with target proteins representing the most complex substrates for enzymatic hydrolysis, namely, containing S-S bonds of the terminal region of your body. In particular, it was shown that when the final product is cleaved from the SUMO-IFN α2 precursor, the disulfide bond is maintained even in the first position of the interferon sequence (RF patent No. 2441072). As for the ability of SUMO to influence the solubility of a hybrid protein, as practice shows, this property significantly depends on the structure of the main partner and the biosynthesis conditions of the fusion protein (Peciak K., 2014; Zhang H., PLoSOne, February 3, 2015). Thus, we can conclude that the formation of the recognition and cleavage region of a protease, which is characterized by a high specificity of hydrolysis, should be considered an absolute advantage of using SUMO in the composition of hybrid precursors.

The obvious disadvantage of a simple combination of SUMO with the protein of interest is that, unlike most other "tags", this protein is not designed for use in the processes of isolation and purification of biosynthesis products. From this point of view, it seems promising to create genetic constructs in which a combination of SUMO is used as auxiliary polypeptides of the "hybrid", which plays the role of a transition region of the cleavage in the predecessor, with other "tags" whose properties (depending on the problem being solved) make it possible to simplify the cleaning and / or shift the balance between insoluble and soluble forms and / or increase the overall yield of the precursor protein (Butt T., 2005; US 7,220,576 05/22/2007; Zhang H. et al., 2015).

Taking into account the fact that hydrolysis with a specific SUMO protease requires not only the presence of a cleavable site, but also the preservation of the native conformation of the protein molecule (T. Butt, 2005), combining this partner with other “tags” usually results in “hybrids” large enough. However, this problem can be partially solved by using the well-known tactics of removing parts that are not functionally significant from the general structure of the precursor. In particular, for this purpose, some researchers use forms of SUMO, "shortened" from the N-terminus to 9-12 amino acids, because according to the current point of view, in order to maintain the necessary conformation of the molecule of this protein and the hydrolysis reaction, the amino acid sequence from 13 to 98 amino acid residues is necessary (Mossessova E., Lima C., 2000, Mol Cell, 5, 865-876).

In view of the foregoing, the closest analogue of the present invention can be considered the development presented in the patent of Russian Federation No. 2453604 and revealing the hybrid protein 10His-SUMO-IFN α2b, where SUMO from Sac. cerevisiae (Smt3) is combined with a tag typically introduced in this type of construct to isolate the fusion protein using metal chelate chromatography; expression vector; recombinant E. coli strain and method for producing a methionine-free target product.

With regard to the prototype, the following disadvantages can be noted.

a) The choice of a “tag” is not good enough, it is used for affinity purification of a hybrid precursor, during which Ni ions (Cu, Zn, Co) are used, which form a coordination bond with the matrix. This determines the possibility of contamination of intermediate products with ions of the named metals and, accordingly, checking the material for their presence, and if detected, their complete removal. Perhaps this circumstance explains that the His tag present in the vector structure often remains unused during cleaning if the latter is carried out on a production scale.

b) Chromatographic purification of products involves 4 stages, one of which is reverse phase chromatography, which requires special equipment, maintenance and materials. In addition, it is known that the organic solvents used in this process, in particular TFA, have a rather severe effect on the protein molecule, which may result in irreversible changes in its conformation. And, finally, the use of reversed-phase chromatography in the preparation of recombinant proteins for medical use appears to be highly undesirable, since it creates additional difficulties in terms of safety (quality) of the preparations obtained, and, at the very least, requires their careful examination for “traces” of toxic impurities First of all, acetonitrile, which is used in very high concentrations (70-80 %%).

c) Incomplete opportunities for removing parts that do not carry a functional load from the hybrid structure.

The objective of the present invention was to develop an expression system (genetic construct and, accordingly, the precursor encoded by it, the expression vector and the E. coli cell transformed by it), providing an unmodified target protein, in particular interferon alpha 2b, in a technologically simple and economical way, guaranteeing lack of contamination of pharmaceutical products with traces of harmful impurities.

Disclosure of invention

The solution to this problem envisaged: a) investigation of the possibility of improving (the largest possible reduction in size without disturbing the proteolytic cleavage process) of the transition region of the hybrid precursor by preparing and testing in the hydrolysis reaction “hybrids” with various “truncations” of the N-terminus of Smt3; b) constructing and expressing recombinant DNA with a sequence encoding the smallest Smt3 fragment selected at stage (a), fused with different kinds of auxiliary sequences with the same target protein, and selecting the optimal (in terms of interest) hybrid constructs; c) the construction and expression of recombinant DNAs with a sequence encoding the Smt3 fragment selected in stage (a), fused with different types of target proteins with the same auxiliary fusion partner, and the selection of optimal (in terms of interest) hybrid constructs; d) testing of the structures selected at stages (b) - (c) in the full cycle of obtaining the target protein encoded by them with the selection, purification and analysis.

a) Previously for SUMO Sac. cerevisiae (Smt3) showed the possibility of shortening the protein from the N-terminus, which does not affect the efficiency of hydrolysis by a specific protease, by 12 amino acids (prototype). However, from an analysis of the three-dimensional structure of the complex of this protein with Ulp1, as well as the sequence alignment pattern of various ubiquitin-like proteins (Mossessova E., 2000, etc.), we made the assumption about the possibility of further truncation of the molecule, which does not entail violation of its conformation. To test this hypothesis, nucleotide sequences optimized for expression in E. coli encoding SUMOΔ19, SUMOΔ23 and SUMOΔ40 were obtained, which, after fusion with a sequence encoding human IFN α2b optimized for expression in E. coli, and introductions into a suitable plasmid were used to transform E. coli cells (Example 1) and biosynthesis of a hybrid precursor. As a result of the analysis of the cleavage of the expressed hybrid proteins by the SUMO protease Ulp1 (Fig. 1), it was shown for the first time that the maximum possible “truncation” of the Smt3 sequence, which does not adversely affect the hydrolysis process, is to remove 23 amino acid residues from the N-terminus.

b) At the second stage, to the selected construct encoding SUMOΔ23-IFN α2b, nuclide sequences encoding various auxiliary proteins were attached from the 5′-end: thioredoxin (TrxA), GST, GST + negatively charged peptide. The choice of this particular group of “tags” was determined by the task: to simplify the cleaning of products and completely eliminate the possibility of their contamination with toxic compounds. After the expression of these design variants in E. coli, it turned out that the inclusion of various fusion partners in the “hybrid” does not affect the efficiency of cleavage of the final product (Example 2, Table 1), as well as the fact that the introduction of GST (in both cases) is essential stimulates the growth of colonies, provides an increase in stable expression and, accordingly, increases the yield of the hybrid protein.

c) Next, an experiment was conducted comparing the cleavage of hybrid proteins of the GST type - (-) peptide-SUMOΔ23-IFN α2b; GST - (-) peptide -SUMOΔ23-caIFN and GST - (-) peptide -SUMOΔ23-proCPB, where caIFN-interferon is a dog, and proCPB is procarboxypeptidase B. The obtained results showed that SUMOΔ23 is effectively recognized by Ulp1 protease in all three cases, t .e. can serve as a cleavage site in the composition of the hybrid precursor of various proteins of interest (example 3). In addition, the results of the analysis allowed us to conclude that the hybrid protein partner GST that we created - (-) peptide - SUMOΔ23 (abbreviated GLSΔ23) creates particularly favorable conditions for the purification of products, because allows you to quickly dissolve the "inclusion body", to clean them on a conventional anion exchanger (Fig. 2) and to easily separate the partner from the target protein (Fig. 3).

To implement the full cycle of obtaining and purification of the unmodified recombinant protein, a construct was selected that encodes the GST precursor - (-) peptide -SUMOΔ23-IFNα2b (GLSΔ23-IFNα2b).

d) Obtaining human interferon alfa-2b included the creation of an expression vector functionally active in E. coli, the transformation of bacterial cells, selection of positive clones and the preparation of a recombinant strain producing a hybrid precursor, culturing the producer under conditions ensuring the formation of a hybrid protein in an insoluble form, its dissolution , refolding, isolation and purification.

In a particular embodiment, the recombinant DNA encoding the GST precursor - (-) peptide -SUMOΔ23-IFN α2b was inserted into the pGEMEX vector, which should not be a limitation of the invention, since it is obvious that in this case another plasmid could also be a carrier vector from those used in practice to obtain recombinant proteins in E. coli, for example, belonging to the pET series.

As a recipient strain in a particular embodiment, E. coli strain BL21 (DE3) was used, which also does not exclude the possibility of using other known laboratory bacterial strains, preferably with reduced protease activity. The resulting recombinant strain of E. coli BL21 (DE3) / pGLSΔ23-IFNA2B was characterized by the following features:

- under microscopy, cells are straight, rod-shaped, gram-negative, non-spore;

- when growing on nutrient agar, the colonies are smooth, round, shiny, gray, the edges of the colonies are even, with a diameter of 1-2 mm;

- the culture grows well on commonly used nutrient media; when grown in liquid media, YT, LB, the culture has the form of a uniform suspension;

- resistant to the antibiotic ampicillin at a concentration of 100 μg / ml.

The cultivation conditions of the obtained producer strain (see Example 5) were selected so that the bulk of the protein was an insoluble form, since under large-scale production conditions it has advantages both in terms of the possibility of obtaining higher yields of the target product, and from the point of view of convenience handling and storing it. At the same time, if necessary, the ratio between the insoluble and soluble forms of the hybrid precursor could be easily shifted in favor of the latter by changing the temperature and aeration conditions, which, as is known, is greatly facilitated by the presence of glutathione-S-transferase in the structure of the “hybrid” ( Harper S. and Speicher D., 2011, Methods Mol Biol., 681, 259-280).

The hybrid protein was dissolved and refolded by conventional methods (Example 5), and one of two options could be used for its separation, the first of which was based on the binding of GSTc to a glutathione-containing sorbent, and the second on the interaction of a negatively charged peptide with a positively charged sorbent. Subsequent purification of the target product (after cleavage of the Ulp1 "hybrid") in both cases involved chromatographic purification on Q and CM sepharose and gel filtration procedures.

As a result (regardless of the variant of "capture" of the hybrid protein), human IFN α2b was obtained with purity and specific activity indices close to the prototype. At the same time, the proposed DNA design and its intended use made it possible to obtain the target product in a technologically simpler and cheaper way than in the prototype, and initially (without additional checks) to ensure that it is not contaminated with traces of toxic compounds, thereby ensuring the achievement of the planned technical results.

Brief Description of the Figures

FIG. 1. The effectiveness of the hydrolysis of hybrid proteins with variants of the truncated protein SUMO.

Electrophoretic separation of the products of Ulp1 hydrolysis in 14% PAAG.

Lanes 1 and 10 are molecular weight markers (14.4; 18.4; 25; 35; 45; 66.2; 116 kD). Lanes 2-3; 4-5; 6-7; 8-9 - a lysate without the addition of Ulp1 and a lysate incubated with Ulp1 for 1 hour in ice upon hydrolysis of the precursors SUMO-IFNα2b, SUMOΔ19-IFNα2b, SUMOΔ23-IFNα2b, SUMOΔ40-IFNα2b, respectively.

FIG. 2. Pre-purification of the unrenatured fusion protein GLSΔ23-caIFN in 6 M urea on an anion exchange resin.

Electrophoretic separation in 10% SDS page.

Lane 1 - molecular weight markers (14.4; 18.4; 25; 35; 45; 66.2; 116 kD).

Lane 2 — Dissolved inclusion bodies of GLSΔ23-caIFN in 8 M urea.

Lane 3 is the same as lane 2, but 5 times less material is applied.

Lane 4 — washing the column with 0.2 M NaCl. Lane 5 — Elution with 0.5 M NaCl. Lane 6 — washout of the fusion protein in the state of aggregation (1 M NaCl).

FIG. 3. Separation of human carboxypeptidase B from the GLSΔ23-CPB fusion protein. Electrophoretic separation in 12% SDS page.

Lane 1 - molecular weight markers (14.4; 18.4; 25; 35; 45; 66.2; 116 kD).

Lane 2 - insoluble protein after dissolution of inclusion bodies.

Lane 3 — soluble protein after dissolution of inclusion bodies.

Lane 4 - Renatured Hybrid Protein.

Lane 5 — Ulp1 hydrolysis of the renatured GLSΔ23-CPB fusion protein.

FIG. 4. Purification of the hybrid protein on Q-Sepharose Fast Flow.

FIG. 5. Chromatographic separation on Fast Flow Q-Sepharose after enzymatic cleavage of the fusion protein.

FIG. 6. Electrophoretic separation in 14% PAAG.

Lane 1 - molecular weight markers (14.4; 18.4; 25; 35; 45; 66.2; 116 kD).

Lane 2 — soluble protein after dissolution of inclusion bodies.

Lane 3 - insoluble protein after dissolution of inclusion bodies.

Lane 4 - Renatured Hybrid Protein.

Lane 5 — GLSΔ23-IFNα2b fusion protein fraction from Fast Flow Q-Sepharose.

Lane 6 - hydrolysis of Ulp1. renatured fusion protein GLSΔ23-IFNα2b.

Lane 7 — IFNα2b fraction from Q-Sepharose Fast Flow.

Lane 8 — GLSΔ23 fraction with Q-Sepharose Fast Flow.

FIG. 7. Purification of IFNα2b on Fast Flow CM-Sepharose.

FIG. 8. Quantification of specific impurities in an IFNα2b sample by HPLC.

FIG. 9. Comparison of the peptide map of the IFN α2b sample with the peptide map of the standard interferon alpha-2b sample.

The implementation of the invention.

Example 1. The study of the effect of shortening the sequence of SUMO (Smt3) on the expression and cleavage of the hybrid precursor.

a) The design of a variant of the synthetic Smt3 gene (SUMO from Saccharomycescerevisiae, NP_010798.1) linked to the mature human interferon-alpha 2b gene (IFNα2b, NP_000596.2) was developed on the basis of the known amino acid sequence using the DNABuilder program (http: // www .innovationsinmedicine.org / software / DNABuilder), guided by information on the composition of codons, strongly expressed genes (stronglyexpressedgenes) of Eshcherichiacoli.

Using the Clone Manager program (Sci-Ed Software, USA), the obtained nucleotide sequence of the gene was checked for the presence of restriction sites used in further work. The found restriction sites were removed by replacing the corresponding nucleotide triplet using the E. coli codon frequency table.

The nucleotide sequence of the SUMO-IFNα2b gene thus compiled, optimized for expression in E. coli cells, was partitioned into oligonucleotides of 50 np in length. using DNA Builder for subsequent assembly of the complete gene by recombinant PCR. In the design of the 5` flanking primer, the NdeI restriction enzyme site was added to its 5`-terminal sequence, and in the design of the 3` flanking primer, the HindIII restriction enzyme site was added. These sites were necessary for the subsequent creation of the expression construct.

PCR amplification was carried out in two stages. An equimolar mixture (5 μM each) of oligonucleotide primers (all but the extreme ones) comprising the gene mutually overlapping by 15-20 nucleotides, 0.2 μM dNTP solution, 1/10 of the reaction buffer 10 and 1 unit was introduced into the reaction. thermostable PfuI DNA polymerase. The primer annealing temperature was calculated by the standard formula:

T _m (° C) = 2 · [N _A + N _T ] + 4 · [N _C + N _G ] -10,

where N _X is the number of corresponding bases. The reaction was carried out as follows: DNA denaturation at 95 ° C, 5 minutes; then 10 cycles: DNA denaturation at 95 ° C, 1 minute; primer annealing at 55 ° C, 1 minute; DNA synthesis at 74 ° C, 2.5 minutes. The second amplification was carried out using the solution of the first reaction and the extreme primers providing the synthesis of the gene with the necessary restriction enzyme sites as the template, according to the following scheme: DNA denaturation at 95 ° C, 5 minutes; then 30 cycles: DNA denaturation at 95 ° C, 1 minute; primer annealing at 55 ° C, 1 minute; DNA synthesis at 74 ° C, 2.5 minutes.

Mix for PCR (50 μl):

5 μl of a 10-fold buffer for PfuI polymerase (Eurogen);

5 μl of reaction mixture 1

0.5 μl of 100 μM Direct-S primer (SEQ ID NO: 1);

0.5 μl of 100 μM Primer Reverse-I (SEQ ID NO: 2);

1 μl of 10 mm dNTP of each type ("Fermentas");

38 μl of deionized water;

0.5 μl of PfuI DNA polymerase (Fermentas).

After amplification, 5 μl of PCR mixture was analyzed by electrophoresis in 1% agarose gel and a homogeneous fragment of 830 bp was detected. The fragment was isolated from the gel using the Min Elute Gel Extraction Kit (Qiagen, Germany) in accordance with the manufacturer's instructions.

b) The plasmid pGEMEX-1 (Promega, USA) was used as a carrier vector. In order to accumulate plasmids, competent strain cells were transformed

proAB lacI^qZΔM15 Tn10 (Tet^r)] (Stratagene, США), затем из полученных ампициллин-устойчивых трансформантов выделяли препараты плазмидной ДНК с использованием набора QIA prep Spin Miniprep Kit (QIAGEN, Германия) в соответствии с инструкцией изготовителя.Escherichiacoli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [

proAB lacI ^q ZΔM15 Tn10 (Tet ^r )] (Stratagene, USA), then plasmid DNA preparations were isolated from the obtained ampicillin-resistant transformants using the QIA prep Spin Miniprep Kit (QIAGEN, Germany) in accordance with the manufacturer's instructions.

Since there are two NdeI sites in plasmid pGEMEX-1, ternary ligation was used. For this, 200 ng of the original plasmid pGEMEX-1 was hydrolyzed with 10 restriction enzymes NdeI and AatII (New England Biolabs, USA) to obtain a DNA fragment of approximately 2000 bp. In addition, 200 ng of GEMEX-1 was hydrolyzed with 10 AatII and HindIII restriction enzymes (New England Biolabs, USA) to obtain a DNA fragment of approximately 1000 bp. 200 ng of the obtained PCR product (complete sequence of the artificial SUMO-IFNα2b gene) was hydrolyzed with 10 restriction enzymes NdeI and HindIII (New England Biolabs, USA). Fragments isolated from agarose gel were pooled and ligated with T4 DNA ligase. The competent ligase mixture was transformed with competent cells of the Escherichiacoli strain XL1-Blue, and then plasmid DNA preparations were isolated from the obtained ampicillin-resistant transformants using the QIA prep Spin Miniprep Kit (QIAGEN, Germany) in accordance with the manufacturer's instructions. The obtained DNA samples were analyzed by co-hydrolysis with restriction enzymes NdeI / HindIII, and "positive" clones containing NdeI / HindIII fragments of 803, 755 and 2347 bp were selected. For mutations, plasmids were analyzed by DNA sequencing. As a result, an expression vector was obtained, designated by us as pGEMEX-1 / SUMO-IFNA2B.

c) Shortening of the SUMO sequence was carried out by PCR. Plasmid pGEMEX-1 / SUMO-IFNA2B was used as a matrix. The reaction was carried out as follows: DNA denaturation at 95 ° C, 5 minutes; then 30 cycles: DNA denaturation at 95 ° C, 1 minute; primer annealing at 52 ° C, 1 minute; DNA synthesis at 74 ° C, 2 minutes. Synthetic oligonucleotides were used as primers: 3`-terminal primer - HSeq (SEQ ID NO: 3) and 5'-terminal primers S20 (SEQ ID NO: 4), S24 (SEQ ID NO: 5), S41 (SEQ ID NO : 6) to obtain, respectively, SUMOΔ19-IFNα2b, SUMOΔ23-IFNα2b, SUMOΔ40-IFNα2b.

After amplification, 5 μl of PCR mixture was analyzed by electrophoresis on a 1% agarose gel and homogeneous fragments of 824, 836, 887 and 938 bp were detected (SUMOΔ19-IFNα2b, SUMOΔ23-IFNα2b and SUMOΔ40-IFNα2b, respectively).

Fragments were isolated from the gel using the Min Elute Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions, digested with restriction enzymes NdeI and HindIII, or ligated with fragments of plasmid pGEMEX-1, as described in b). Selected plasmids were designated as pGEMEX-1 / SUMOΔ19-IFNA2B, pGEMEX-1 / SUMOΔ23-IFNA2B and pGEMEX-1 / SUMOΔ40-IFNA2B, respectively.

d) Since the genes of the hybrid proteins in the obtained recombinant plasmids in the pGEMEX-1 vector are under transcriptional control of T7 polymerase, E. coli strain BL21 (DE3) [F-, ompT, hsdSB (rB-, mB-), dcm was used for transformation , gal, λ (DE3)], which synthesizes RNA polymerase of the phage T7, and also has a reduced protease activity.

For selection, transformed bacteria were plated on YT agar culture medium containing ampicillin at a concentration of 100 μg / ml. Overgrown bacterial colonies were inoculated with 50 ml of TB5052 culture medium (Studier F.W. Protein Expr Purif. 2005; 41: 207-234) and cultured for 48 hours at 15 ° C, then the bacteria were collected by centrifugation, the supernatant was decanted and the precipitate was frozen at -20 ° C.

To analyze the efficiency of the hydrolysis of hybrid proteins by Ulp1 hydrolase, the cells were thawed and destroyed by ultrasound with periodic cooling in ice in a lysis buffer containing 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1 mM PMSF, pH 9.0. Cell debris was removed by centrifugation at 13,000 rpm for 10 minutes. Ulp1, prepared as described in (Reverter D., Lima C. D. Methods Mol Biol. 2009; 497: 225-239), was added to the supernatant to a final concentration of 2 μg / ml and incubated for 1 hour in ice. The hydrolysis efficiency was analyzed by SDS-PAGE electrophoresis.

As a result of the analysis, it was found that hybrids containing truncated forms of SUMOΔ19 and SUMOΔ23 are hydrolyzed by Ulp1 with the same efficiency as the hybrid with full-sized SUMO, whereas the precursor with SUMOΔ40 does not undergo enzymatic cleavage at all (Fig. 1). For further work, the design of SUMOΔ23-IFNα2bc was selected, which we showed as much as possible shortening SUMO Sac. cerevisiae.

SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 3

SEQ ID NO: 4

SEQ ID NO: 5

SEQ ID NO: 6

Example 2. The study of the effect of various auxiliary polypeptides on the expression and cleavage of the hybrid precursor.

The nucleotide sequences encoding various proteins: thioredoxin (TrxA), GST, GST + negatively charged peptide were attached from the 5′-end of the selected construct in the previous example to the encoding SUMOΔ23-IFN α2b. The choice of this particular group of “tags” was determined by the task: to simplify the cleaning of products and completely eliminate the possibility of contamination with toxic compounds.

a) TrxA

The thioredoxin gene of E. coli tvxA (AAC40210.1) was cloned by PCR using the commercial plasmid pET32a as a template. When designing the 5 ′ Trx-Dir flanking primer (SEQ ID NO: 7), the NdeI restriction enzyme site was added to its 5`-terminal sequence, and when designing the Trx-Rev 3 ′ flanking primer (SEQ ID NO: 8), the EcoRI restriction enzyme site . These sites were necessary for subsequent cloning into the pGEMEX-1 plasmid (for pET plasmids, the NcoI site is used instead of NdeI). Cloning of the PCR product was performed as described in Example 1 at the restriction sites NdeI and EcoRI. The resulting plasmid pGEMEX-1 / TrxA was analyzed by DNA sequencing within the insert.

To assemble the final expression plasmid vector, the EcoRI site was introduced before the SUMOΔ23-IFNα2b hybrid gene sequence using the PCR method using pGEMEX-1 / SUMOΔ23-IFNA2B plasmid as a template. An EcoRI site was provided in the sequence of the 5`-terminal primer E-S24 (SEQ ID NO: 9), HSeq (SEQ ID NO: 3) was used as the 3`-terminal primer. The PCR product was cloned into the vector pGEMEX-1 / TrxA as described in Example 1 at EcoRI and HindIII restriction sites. The resulting plasmid pGEMEX-1 / TrxA-SUMOΔ23-IFNA2B was analyzed by DNA sequencing within the insert.

b) GST

The glistathione S transferase gene from Schistosomajaponicum (GST) was cloned from the commercial vector pET28GST-LIC (GenBank: EF456739.1) by PCR using primers: 5`-flanking GST-Dir (SEQ ID NO: 10) containing the NdeI restriction site , and the 3`-flanking primer GST-Rev (SEQ ID NO: 11) containing the EcoRI restriction site. The PCR product was cloned into the plasmid pGEMEX-1 as described in Example 1 at the NdeI / EcoRI restriction sites. The resulting plasmid pGEMEX-1 / GST was analyzed by DNA sequencing within the insert and used to obtain the plasmid pGEMEX-1 / GST-SUMOΔ23-IFNA2B similar to the plasmid pGEMEX-1 / TrxA-SUMOΔ23-IFNA2B described above.

C) GSTc (-) peptide (Linker)

The nucleotide sequence encoding the negatively charged peptide was adapted into the pGEMEX-1 / GST-SUMOΔ23-IFNA2B plasmid between the GST and SUMOΔ23 genes, similarly to the preparation of the pGEMEX-1 / GST-SUMOΔ23-IFNA2B plasmid, where a synthetic oligonucleotide was used as a 3` flanking linker SEQ ID NO: 12. The result was a plasmid, which was designated pGEMEX-1 / GLSΔ23-IFNA2B.

SEQ ID NO: 7

SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 10

SEQ ID NO: 11

SEQ ID NO: 12

Linker:

d) Expression analysis and analysis of the hydrolysis of hybrid proteins Ulp1.

The obtained expression vectors transformed E. coli cells of strain BL21 (DE3), seeded on selection agar medium YT containing ampicillin. The grown bacterial colonies were inoculated with ampicillin-containing TB5052 liquid medium and cultured for 16 hours at 37 ° C or 48 hours at 15 ° C. Then the bacteria were collected by centrifugation, the supernatant was decanted and the precipitate was frozen at -20 ° C.

To analyze the efficiency of the hydrolysis of hybrid proteins by Ulp1 hydrolase, the cells were thawed and sonicated with periodic cooling in ice in a buffer containing 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1 mM PMSF, pH 9.0. Cell debris was removed by centrifugation at 13,000 rpm for 10 minutes. ULP1 was added to the supernatant to a final concentration of 2 μg / ml and incubated for 1 hour in ice. The hydrolysis efficiency was analyzed by SDS-PAGE electrophoresis. The data are presented in table 1.

The results obtained indicate that in all investigated variants of the precursor containing various “tags” in their structure, the normal process of hydrolysis of Ulp1 is observed. At the same time, it should be noted that the presence of GST in the composition of the hybrid protein (see the last two lines in the table) positively affects the growth of colonies, respectively, the overall yield of the hybrid protein.

Example 3. Obtaining and testing DNA constructs of the proposed type for various target proteins.

In addition to human interferon, dog interferon and human procarboxypeptidase B were used as target proteins in the proposed precursor proteins.

a) Interferon dogs (caIFN)

The SUMOΔ23-caIFN fusion protein gene, consisting of the truncated SUMOΔ23 gene (Example 2) and the dog alpha interferon gene alpha 1 (ABF68838.1), was assembled using the PCR method from synthetic oligonucleotides based on the known amino acid sequence. When designing the sequences, they were guided by information on the frequency of occurrence of codons in strongly expressed Eshcherichiacoli genes. Flanking primers 5`-E-S24 (SEQ ID NO: 9) and Ca-Rev (SEQ ID NO: 13) contained restriction enzyme sites EcoRI and HindIII, respectively. These sites were necessary to obtain the expression construct pGEMEX-1 / GLSΔ23-caINF. The assembly of the construct was carried out similarly to the assembly of the vector pGEMEX-1 / GLSΔ23-IFNA2B (the designs differ only in that the dog gene interferon alpha 1 is included in the construct instead of the human interferon alpha-2b gene).

b) Procarboxypeptidase (proCPB)

The human procarboxypeptidase B gene (NP_001862.2) was assembled by PCR and adapted to the pGEMEX-1 / GLSΔ23-proCPB plasmid vector similar to the previous examples using the 3`-flanking CPB-Rev primer (SEQ ID NO: 14).

SEQ ID NO: 13

SEQ ID NO: 14

c) E. coli BL21 (DE3) cells were transformed with constructs, recombinant strains were cultured at 37 ° C to obtain the precursor in insoluble form, followed by dissolution of inclusion bodies, refolding of the hybrid protein and hydrolysis of its Ulp1 (all of these processes are described in more detail in Example 5 )

The data obtained allowed us to conclude that the hybrid partner protein GLSΔ23 that we created allows us to easily dissolve the inclusion bodies, successfully clean them on the anion exchange resin (Fig. 2) and separate it from the target protein (Fig. 3). Moreover, SUMOΔ23 was efficiently cleaved by Ulp1 protease in all three cases, i.e. served as a cleavage site in the hybrid precursor of various proteins of interest.

Example 4. Obtaining a recombinant strain producing IFN alpha alpha2b person.

The recombinant strain producing human IFN alpha2b was obtained based on the primary culture of E. coli BL21 (DE3) cells transformed with plasmid pGLSΔ23-IFNA2B described in Example 3 after 3-fold reseeding.

The resulting recombinant strain of E. coli BL21 (DE3) / pGLSΔ23-IFNA2B was characterized by the following features:

- under microscopy, cells are straight, rod-shaped, gram-negative, non-spore;

- when growing on nutrient agar, the colonies are smooth, round, shiny, gray, the edges of the colonies are even, with a diameter of 1-2 mm;

- the culture grows well on commonly used nutrient media; when grown in liquid media, YT, LB, the culture has the form of a uniform suspension;

- resistant to the antibiotic ampicillin at a concentration of 100 μg / ml.

Example 5. Obtaining unmodified IFN human alpha2b using recombinant E. coli strain BL21 (DE3) / pGLSΔ23-IFNA2B

a) Cultivation.

The cultivation of the producer strain of the hybrid precursor of human interferon alfa2b was carried out on a laboratory fermenter with a working volume of 3 l in TB medium (yeast extract - 24 g / l, tryptone - 12 g / l, potassium phosphate monosubstituted - 2.31 g / l, potassium phosphate disubstituted - 12.54 g / l, glycerin - 4 ml / l). Cultivation was carried out at a temperature of 37 ° C and maintaining dissolved oxygen at a level of at least 10%. Aeration was maintained at a constant level of 1 rpm / min. The stirrer speed was automatically adjusted in the range of 300-800 rpm depending on the data of the dissolved oxygen sensor. The synthesis of the target protein was induced when the culture reached an optical density of 4 p.u. by adding IPTG to a final concentration of 0.2 mM. Cultivation was continued until the culture reached a stationary growth phase. Estimation of the yield of the target protein was determined by electrophoresis in SDS page according to the Lamley method. At the fermentation stage, the yield of the hybrid protein was estimated to be at least 1000 mg / L.

b) Isolation, purification and enzymatic hydrolysis of the hybrid protein.

- getting Taurus inclusion.

To obtain inclusion bodies, the biomass of the producer strain was suspended in the composition buffer: 50 mM Tris-HCl, 100 mM sodium chloride, 5 mM EDTA, 0.1 mM PMSF, pH 8.0 in a ratio of 1:10 and twice passed through a high-pressure disintegrator at a pressure of 850 bar.

- dissolution of inclusion bodies.

Insoluble inclusion bodies were isolated, purified and dissolved in buffer (8 M urea, 100 mM Tris-HCl pH 9.5, 2 mM EDTA, 20 mM 2-mercaptoethanol). The resulting mixture was incubated at room temperature for 6 hours. Insoluble material was removed by centrifugation (15000 g), and the solution was filtered through a membrane with a pore diameter of 0.45 μm.

- renaturation of the hybrid protein.

The GST-SUMOΔ23-IFN α2b protein was renaturated by diluting the resulting solution 20 times with 20 mM glycine pH 9.3, 0.4 M urea, 0.1% Tween 20.1 mM cysteine, 0.1 mM cystine. The reaction mixture was incubated for 18 hours at a temperature of + 8 ÷ 12 ° C and constant stirring at a speed of 120 rpm.

- purification of the hybrid protein

Since the GST tag and the charged linker are part of the fusion protein, fusion protein purification could be carried out by one of the two options described below.

Option 1.

After renaturation, 10% phosphoric acid was added to the reaction mixture with stirring at a speed of 120 rpm until a pH of 7.3 was established and filtered through a membrane with a pore diameter of 0.45 μm. The resulting solution was purified on an affinity sorbent.

The hybrid protein capture procedure was performed using an XK 50/20 column (GE Healthcare) with a 200 ml working volume containing the Glutathione Sepharose 4 B sorbent (GE Healthcare) at a working flow rate of 7 ml / min. First, the column was equilibrated with 400 ml of PBS pH 7.3 buffer, then a hybrid protein solution was applied. After that, the column was washed with 400 ml of PBS pH 7.3. The fusion protein was eluted with 800 ml of A1 buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). Elution GST-SUMOΔ23-IFN α2b was monitored by optical density at a wavelength of 280 nm. The eluate was subjected to enzymatic hydrolysis (see the corresponding section).

Option 2

The solution with the renatured protein was filtered through a membrane with a pore diameter of 0.45 μm. The resulting hybrid protein solution was purified on an anion exchange sorbent.

The hybrid protein capture procedure was performed using an XK 26/20 column (GE Healthcare) with a working volume of 70 ml containing Fast Flow Q-Sepharose sorbent (GE Healthcare) at a working flow rate of 7 ml / min. The column was pre-equilibrated with 200 ml of buffer A2 (50 mM Tris-HCl pH 8.0), then a protein solution was applied. After that, the column was washed with 350 ml of buffer A2. The hybrid protein was eluted using a linear gradient of 0-50% B1 buffer (50 mM Tris-HCl, 1 M sodium chloride, pH 8.0) in a volume of 700 ml. The elution of the hybrid precursor was monitored by optical density at a wavelength of 280 nm (Fig. 4). The eluate was subjected to enzymatic hydrolysis (see below).

- enzymatic cleavage of the hybrid protein by SUMO protease (Ulp1).

Enzymatic cleavage of the protein GST-SUMOΔ23-IFNα2b, isolated by any of the above options, was carried out with stirring at a speed of 120 rpm for 1.5 hours at a temperature of + 20 ÷ 25 ° C. For this, Ulp1 proteinase was added to the solution of the hybrid protein at the rate of 2 mg proteinase (Reverter D., et al., 2009) per 1 g of the hybrid protein. In the case of option 2, the solution of the hybrid protein was previously diluted 5 times with 50 mM Tris-HCl pH 8.0 buffer before the enzyme was added.

Upon completion of the enzymatic digestion, the resulting solution was filtered through a membrane filter with a pore diameter of 0.22 μm and subjected to further chromatographic purification.

c) Purification of the target protein (IFN alpha2b)

- chromatography on Q-Sepharose.

An XK 26/20 column (GE Healthcare) was used with a working volume of 70 ml containing Fast Flow Q-Sepharose sorbent (GE Healthcare) at a working flow rate of 7 ml / min. After equilibrating the column with 200 ml of A2 buffer (50 mM Tris-HCl pH 8.0), the filtrate obtained in the previous step was applied. After this, the column was washed with 300 ml of buffer A2. The target protein was eluted with a linear gradient of 0-50% B1 buffer (50 mM Tris-HCl, 1 M sodium chloride, pH 8.0) in a volume of 700 ml. Elution of IFNα2b was monitored by optical density at a wavelength of 280 nm (Fig. 5, 6). The fractions were collected in 30 ml fractions in polypropylene tubes.

The collected eluatase fractions were oxidized, for which they were combined and, with stirring, at a speed of 120 rpm, was diluted 5 times with A3 buffer (50 mM sodium acetate, pH 4.5). The resulting solution was filtered through a 0.22 μm filter.

- chromatography on CM-Sepharose.

An XK 26/20 column (GE Healthcare) with a working volume of 50 ml containing a Fast Flow CM-Sepharose sorbent (GE Healthcare) was used at a working flow rate of 5 ml / min. The column was equilibrated with 150 ml of A3 buffer, a protein solution was applied, then washed with 250 ml of A4 buffer (50 mM sodium acetate, 0.1 M sodium chloride, 0.1% Tween 20, pH 4.5) and 200 ml of A3 buffer. The target protein was eluted with a linear gradient of 0-50% B2 buffer (50 mM sodium acetate, pH 4.5, 1 M sodium chloride) in a volume of 500 ml. IFNα2b elution was monitored by optical density at a wavelength of 280 nm (FIG. 7). Upon reaching 100 mUU, 30 ml fractions were collected in polypropylene tubes.

The collected eluate fractions were analyzed by HPLC. Fractions containing the target protein with a purity of at least 96% were combined and passed through a membrane filter with a pore diameter of 0.22 μm.

- gel filtration.

Gel filtration was performed on a column (GE Healthcare) with a working volume of 2000 ml containing a Sephadex G-50 sorbent (GE Healthcare), at a working flow rate of 4 ml / min. The column was pre-equilibrated with 3 L of A5 buffer (20 mM sodium acetate, 150 mM sodium chloride, pH 5.0), after which the solution obtained after purification on CM-Sepharose was applied, and the column was washed with 2 L of A5 buffer. The elution of the target protein was monitored by optical density at a wavelength of 280 nm. Upon reaching an OD value of 50 mUU, fractions were collected in polypropylene tubes.

The collected eluate fractions were analyzed by HPLC. Fractions containing unmodified interferon alpha-2b with a purity of not less than 96% were combined and passed through a sterile membrane filter with a pore diameter of 0.22 μm.

d) The yield and characteristics of the target product.

In the case of using the first variant of purification of the hybrid precursor based on affinity affinity, the yield of the target protein was more than 100 mg per 1 liter of culture, while the use of the second variant, using the presence of a negatively charged peptide in the hybrid precursor, made it possible to increase the yield of the target protein by 1.5 times , and get more than 150 mg with 1 liter of culture (see table 2).

Purified human interferon alpha-2b obtained using both option 1 and option 2 isolation of the hybrid precursor has the following characteristics:

- purity not lower than 96%, (Fig. 8);

- the peptide map of the obtained protein basically corresponds to the peptide map of a standard sample of interferon alpha-2b (Interferonalpha-2bCRS, Councilof Europe; European Directorate for the Quality of Medicines European Pharmacopoeia; Strasbourg, cat. No. 10320301), (Fig. 9);

- the specific activity determined by the biological method using a culture of MDBK cells and vesicular stomatitis virus (VSV) according to (European Pharmacopoeia 6.0, Interferonalfa-2 concentrated solution) is at least 1.4 × 10 ⁸ IU / mg.

Thus, in the implementation of the present invention was obtained IFN α2b person with indicators of purity and specific activity close to the prototype. Moreover, the proposed DNA construct and its intended use allowed to obtain the target product in a technologically simpler and cheaper (without the use of reverse phase chromatography) method than in the prototype, and initially (without additional checks) to ensure that it was not contaminated with toxic traces compounds.

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Claims (7)

1. An element of a genetic construct that encodes a hybrid precursor of an unmodified form of the target recombinant protein, where the named element is a polynucleotide with a nucleotide sequence encoding SUMO Sac protein shortened from the N-terminus to 23 amino acid residues. cerevisiae (Smt3), and in the hybrid precursor plays the role of the recognition and cleavage region of the specific protease Ulp1. 2. The genetic construct encoding the hybrid precursor of the unmodified form of the target recombinant protein, which consists of operatively linked nucleotide sequences encoding an auxiliary amino acid sequence, the amino acid sequence of the recognition and cleavage region of a specific protease and the sequence of the target protein, where the specified recognition and cleavage region is encoded by an element according to . one 3. The genetic construct encoding the hybrid precursor of the unmodified form of the target recombinant protein, which consists of operatively linked nucleotide sequences encoding an auxiliary amino acid sequence, the amino acid sequence of the recognition and cleavage region of a specific protease and the sequence of the target protein, where the specified recognition and cleavage region is encoded by an element according to . 1, and the auxiliary amino acid sequence is ene glutathione-S-transferase (GST), related to the charged region of the cleavage peptide linker. 4. The genetic construct encoding the hybrid precursor of the unmodified form of the target recombinant protein, which consists of operatively linked nucleotide sequences encoding the GST amino acid sequence, a negatively charged peptide linker, shortened from the N-terminus to 23 amino acid residues of the SUMO Sac protein. cerevisiae (Smt3) and human interferon alpha-2b sequence 5. Recombinant expression vector functionally active in E. coli containing, under the control of a promoter, the genetic construct of claim 4. 6. The culture of E. coli cells transformed with the expression vector according to claim 5 and providing the synthesis of a hybrid precursor encoded by the genetic construct according to claim 4. 7. A method for producing recombinant human interferon alfa-2b, comprising cultivating a producer of the interferon alfa-2b hybrid precursor under conditions ensuring the formation of the product in insoluble form, separation and dissolution of inclusion bodies, refolding of the hybrid protein, its isolation and purification, hydrolysis with SUMO protease (Ulp1) and chromatographic purification of the target protein, characterized in that the E. coli cell culture described in clause 6 is used as producer for isolation and purification of the hybrid precursor Tween calves use affinity chromatography on glutathione-Sepharose 4B or ion-exchange chromatography on Q-Sepharose, and two stages of ion-exchange chromatography, Q-Sepharose and CM-Sepharose, are sequentially used to purify the target protein.

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