RU2453604C1 - Hybrid protein (versions), bacterial strain escherichia coli - hybrid protein producer (versions) and method for producing methionine-free human interferon alpha-2 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: constructing a recombinant plasmid DNA containing structural gene of human interferon alpha-2b or alpha-2a under control of regulated promoter enables producing bacterial strains Escherichia coli providing synthesis of insoluble precursor proteins of mature methionine-free human interferons alpha-2b or alpha-2a. The precursor proteins of the sequence of the mature methionine-free human interferons alpha-2b or alpha-2a are fused with a sequence of SUMO (SMT3) protein of yeast Saccharomyces cerevisiae. What is produced is the bacterial strain Escherichia coli providing biosynthesis of ULP275 proteinase for enzymatic processing of the precursor protein of methionine-free interferons. What is developed is a method for producing methionine-free interferons which provides carrying out the coupled renaturation processes, disulphide link formation and enzymatic segregation of methionine-free human interferons alpha-2b or alpha-2a from the precursor protein under ULP275 proteinase.

EFFECT: method improvement.

6 cl, 20 ex

Description

The invention relates to the field of biotechnology and medicine, and relates to a method for producing methionine-free (not containing N-terminal methionine) human interferon alpha-2.

Two allelic variants of interferon are attributed to human interferon alpha-2 (IFN-α2): interferon alpha-2b and interferon alpha-2a, which differ in the coding region by a single nucleotide substitution at position 137 (2a: A, 2b: G). [Kaluz et al., 1993 Acta Virol. 37: 97-100; Kaluz et al., 1994, Acta Virol. 38: 101-4; Lee et al., 1995, J Interferon Cytokine Res. 15: 341-9], which leads to amino acid substitution (2a: Lys, 2b: Arg).

Human interferons alpha-2 belong to the class of cytokines,

possessing antiviral activity, and are representatives of the alpha interferon family, secreted by almost all types of virus-infected human cells [Pfeffer et al., 1998, Cancer Research 58: 2489-2499].

Recombinant IFN-α2 is used to treat viral and tumor diseases [Samuel, 2001, Clinical Microbiology Reviews, 14: 778-809; Schadendorf et al., 2009, Annals of Oncology, 20 (Supplement 6): vi41-vi50; Pfeffer et al., 1998, Cancer Research 58: 2489-2499], including for the treatment of solid tumors such as bladder cancer, kidney cancer, HIV-induced Kaposi's sarcoma and others [Torti, 1988, J. Clin. Oncol., 6: 476-483; Vugrin et al., 1985, Cancer Treat. Rep., 69: 817-820; Rios et al., 1985, J. Clin. Oncol., 3: 506-512]. IFN-α2 are the main therapeutic agents used to treat chronic forms of hepatitis B and C [Clark V., Nelson D.R., 2009, Clin Liver Dis, 13: 351-363].

The N-terminal amino acid residue of naturally occurring IFN-α2 is cysteine linked by a disulfide bond to 98 protein cysteine [Bodo G., Fogy I., 1985, The Interferon System, pp.23-27; Wetzel et al., 1981, J. Interfer. Res., 1: 381-90]. At the same time, some of the recombinant IFN-α2 molecules isolated from Escherichia coli (E. coli) cells have an additional methionine (formylmethionine) residue at the N-terminus, which makes recombinant IFN-α2 a modified protein.

Excess N-terminal methionine can affect the stability and immunogenicity of proteins [Ben-Bassat A., Bauer K., 1987, Nature 326, 315], and also in some cases leads to the loss of their biological activity [Rabbani et al., 1988, J Biol Chem, 263: 1307-1313]. Due to the fact that since 2005 the European Pharmacopoeia has limited the production and use in Europe of drugs containing modified IFN-α2 [Eur. Pharm., 5 ^th ed .: 2004], the problem of removing the N-terminal methionine from the composition of IFN-α2 of bacterial origin has become even more important.

In E. coli cells, which remain one of the main producers of recombinant proteins, the removal (processing) of the N-terminal methionine is determined by the radius of the side radical of the amino acid residue of the synthesized protein following methionine [Hirel et al., 1989, Proc Nati Acad Sci USA, 86 (21 ): 8247-51; Dalb⌀ge et al., FEBS Lett. 1990; 266 (1-2): 1-3]. However, such processing is often incomplete in the case of protein overexpression [Yasueda et al., 1991, Appl Microbiol Biotechnol, 36: 211-215; Hwang et al., 1999, Biochem J, 338: 335-342].

Among the enzymatic methods for removing the N-terminal methionine from the composition of recombinant proteins include:

- removal of methionine using methionine aminopeptidase [Shapiro et al., 1988, Anal. Biochem 175: 450-461; Solbiati et al., 1999, J Mol Biol, 290: 607-14; Liao et al., 2004, Protein Science, 13: 1802-1810];

- methods based on the biosynthesis of an elongated protein, between the N-terminal methionine and the mature part of which a short sequence of a proteinase recognition site is introduced. Subsequent proteinase treatment of the elongated protein removes the introduced sequence along with the N-terminal methionine [US 5665566; Belagaje et al., 1997, Protein Sci, 6: 1953-1962; Hosfield, T., Lu Q., 1999, Anal Biochem, 269: 10-6; Jenny R.J. et al., 2003, Protein Express Purif, 31: 1-11].

The inventive method is based on the biosynthesis of E. coli cells of precursors of non-methionine human alpha-2 interferons containing sequences of mature interferons fused to the sequence of the yeast protein SUMO (Small Ubiquitine-like protein Modifier) [Johnson et al., 1997, EMBO J 16: 5509-5519; Muller et al., 1998, EMBO J, 17: 61-70], belonging to the ubiquitin-like protein family (UPB).

UPB are labels of a protein nature, which are used in the eukaryotic cells to label other proteins, as a result of which the latter change their functional activity [Hochstrasser M., 2000, Science, 289: 563-564]. In addition to ubiquitin, the UPB family includes SUMO, Rub1, Hub1, ISG15, Apg12, Apg8, Urm1, Ana 1a and Ana 1b proteins. During labeling, the C-terminal residues of UPB are conjugated to the ε-amino groups of lysine on the surface of the modified proteins [Jentsch S., Pyrowolakis G., 2000, Trends Cell Biol, 10: 335-342; Muller et al., 2001, Nat Rev Mol Cell Biol 2: 202-210]. The majority of biotechnological methods for using the UPB family of proteins, with the exception of APG12 and URM1, are based on their natural ability to be synthesized in the form of elongated precursors undergoing post-translational processing, leading to the release of the universal G-Gly C-terminal sequence [Malakhov et al., 2004 , J Struct Funct Genom, 5: 75-86].

The yeast genome Saccharomyces cerevisiae (S. cerevisiae) contains the SMT3 gene located between the nucleotide positions 1469403-1469708 of chromosome IV [NCBI database]. The SMT3 gene encodes a protein SMT3p - yeast SUMO. Yeast SUMO is synthesized as a 101-amino acid precursor, of which 98 are mature proteins, of which residues 13-98 are important for the formation of its native structure [Mossessova E., Lima C. D., 2000, Mol Cell, 5: 865-876 ]. Eukaryotic SUMOs, including yeast SMT3p, have a similar three-dimensional structure, which is similar to that of ubiquitin and is characterized by tightly laid beta layers wrapped around a single alpha helix [Bayer et al., 1998, J Mol Biol, 280: 275-286]. Like other members of the UPB family, SUMO proteins from various organisms are synthesized as precursors containing C-terminal extensions of 2 to 12 amino acid residues that are a continuation of the conservative C-terminal Gly-Gly motif [Muller et al., 2001, Nature , 2: 202-210]. Despite various C-terminal lengthenings and a rather low degree of homology of amino acid sequences, for example, human yeast SUMO and SUMO-1 (47%), S. cerevisiae yeast proteinase Ulp1 is able to process both protein precursors [Mossessova E., Lima C.D. , 2000, Mol. Cell, 5: 865-876].

Proteinase Ulp1 belongs to the ULP cysteine proteinase family. The catalytic domain of Ulp1 proteinase is localized in the C-terminal region of the protein; in particular, its C-terminal fragment of 275 amino acid residues has full enzymatic activity [Li S.-J., Hochstrasser M., 2003, J Cell Biol, 160: 1069-1081 ; Butt et al., 2005, Protein Express Purif, 43: 1-9] (hereinafter, ULP275 proteinase). Like other members of this family in vivo, Ulp1 proteinase deconjugates SUMO and labeled target proteins and is also responsible for processing the native SUMO precursor. The main best-known features of Ulp1 proteinase are two: 1) the action of Ulp1 proteinase is directed to the hydrolysis of peptide or isopeptide bonds connecting the conserved C-terminal Gly-Gly dipeptide of mature SUMO and the amino acid residue of the target protein; 2) by binding to the target protein, the ULP family proteinases recognize not a separate site, but the three-dimensional structure of SUMO proteins [Butt et al., 2005, Protein Express Purif, 43: 1-9; Wilkinson, 1997, FASEB J, 11: 1245-1256; Chang C. H., Back S. H., 1999, Biochem Biophys Res Commun, 266: 633-640].

The property of the native Ulp1 proteinase and its C-terminal fragment is to recognize the SUMO protein and its derivatives as part of conjugates and precursors and to carry out their highly specific and efficient processing almost independently of the nature of the amino acid residue (excluding proline) connected to the conserved C-terminal Gly-Gly dipeptide, underlies the use of artificially created expression SUMO systems. Their distinguishing feature is the biosynthesis of target proteins in the form of hybrid precursors containing in the N-terminal region a sequence of SUMO protein or its derivatives, deleted, for example, during proteolytic processing in vitro [Malakhov et al., 2004, J StructFunctGenom, 5: 75- 86; Marblestone et al., 2006, Protein Science, 15: 182-189]. SUMO systems are used to: (1) increase the expression of proteins in cells of eukaryotic and prokaryotic organisms; (2) increasing the solubility of expressed proteins; (3) facilitating the process of protein purification due to the inclusion of affinity N-terminal polypeptides in their composition; (4) obtaining proteins with altered N-terminal residues [Malakhov et al., 2004, J Struct Funct Genom, 5: 75-86].

A fusion protein is known consisting of yeast SUMO protein and human IkB protein [US 6872551], which is a recombinant product, it is purified from E. coli cell lysates and digested with Ulp1 proteinase. However, unlike IFN-α2, the IkB protein at the junction with SUMO does not have a cysteine residue forming a disulfide bond. Thus, Ulp1-processing of the SUMO-IkB hybrid results in the release of the usual amino-terminal amino acid residue of the IkB protein, rather than a strong structural element containing a disulfide bond, as is required in the case of the native non-methionine IFN-α2. For this reason, the data on the processing efficiency of hybrid proteins like SUMO-IkB could not guarantee that Ulp1 processing will be effective also for SUMO hybrids and native non-methionine IFN-α2. No information was found on the biosynthesis of IFN-α2 fusion proteins with SUMO protein in information sources.

Thus, the properties of the Ulp1 SUMO proteinase system described above can be summarized as follows: 1) it is known that Ulp1 proteinase is capable of hydrolyzing the peptide bond connecting the conserved C-terminal Gly-Gly dipeptide of mature SUMO with any subsequent amino acid residue (except proline), which in turn, may be linked by a single peptide bond to the following amino acid residue; 2) it is known that Ulp1 proteinase is able to hydrolyze an isopeptide bond connecting the conserved C-terminal Gly-Gly dipeptide of mature SUMO with an amino acid residue of a protein (lysine), which in turn can be linked by two peptide bonds to neighboring amino acid residues of the protein; 3) it was not known whether Ulp1 proteinase is able to hydrolyze a peptide bond connecting the conserved C-terminal Gly-Gly dipeptide of mature SUMO with the amino acid residue of the protein (cysteine), which is linked to other amino acid residues of the protein by both peptide and disulfide bonds, i.e. is part of an unusual structural element of the protein. However, it was precisely this Ulp1 proteinase activity that was necessary for the hydrolysis of the peptide bond between the C-terminal dipeptide of SUMO and the N-terminal cysteine of native human IFN-α2.

The closest analogue of the claimed strains is a recombinant strain of E. coli KCCM 10053 - producer of human interferon alpha-2b containing N-terminal methionine [KR 20000065580].

The closest analogue of the proposed method is the method [KR 20000065580] for the preparation of human bezmethionine-interferon alpha-2b by digestion with methionine aminopeptidase from the composition of the precursor protein methionine-containing interferon synthesized in cells of E. coli strain KCCM 10053. The main disadvantage of the method is the closest analogue is the need for a long (12-20 hours) enzymatic treatment of the protein precursor. Further difficulties may be caused by the subsequent conversion of the methionine-free protein to a biologically active state under the conditions necessary for the formation of disulfide bonds [Morehead et al., 1984, Biochemistry, 23: 2500-7], as well as the process of separation of the target methionine-free interferon from the methionine-containing precursor, due to their slight physico-chemical differences.

The task of the claimed group of inventions is to expand the arsenal of methods for producing human methionine-free IFN-α2.

The problem is solved by constructing:

- a hybrid protein - the precursor of methionine-free interferon alpha-2, an integral part of which is the sequence of human methionine-free interferon alpha-2b or alpha-2a fused to the SUMO protein sequence of the yeast Saccharomyces cerevisiae;

- strain of bacteria Escherichia coli VKPM B-10414 - producer of the precursor of methionine-free interferon alpha-2b person;

- strain of bacteria Escherichia coli VKPM B-10700 - producer of the precursor of bezmethionine interferon alpha-2a person

 and development

- a method for producing human methionine-free interferon alpha-2 by microbiological synthesis and isolation of a hybrid protein-precursor of methionine-free interferon alpha-2b or alpha-2a, an integral part of which is a sequence of human methionine-free interferon alpha-2b or alpha-2a, fused to the sequence of yeast SUMO protein Saccharomyces cerevisiae, followed by enzymatic cleavage of its composition of methionine-free interferon by treatment with proteinase from the ULP family under conditions conducive to the formation of interferon correctly closed disulfide bonds.

The SUMIN and SUMIN-2a fusion proteins represent the principal parts of the HSI and HSI-2a fusion proteins, the precursors of the non-methionine human alpha-2b and alpha-2a interferons, respectively, while the N-terminal extensions of the SUMIN and SUMIN-2a fusion proteins are present hybrid proteins HSI and HSI-2a, perform only an auxiliary role and can be used, for example, at separate stages of protein purification.

The inventive method is based on the biosynthesis of hybrid proteins HSI and HSI-2a, the precursors of the methionine-free interferons alpha-2b and alpha-2a, containing in their composition sequences of the fusion proteins SUMIN and SUMIN-2a, respectively, and the subsequent release of methionine-free interferons alpha-2b and alpha -2a from the composition of hybrid proteins during proteolytic processing using ULP275 proteinase.

The proposed method is based on the fact established by the authors that, in contrast to proteinases, the activity of which is inhibited by a structural element of a protein containing a disulfide bond located near the processing site [Hubbard et al., 1994, Protein Sci, 3: 757-768 ; Hubbard et al., 1998, Protein Engineering, 11: 349-359], ULP275 proteinase is able to efficiently hydrolyze a peptide bond near such a structural element. This ULP275 proteinase activity, which we have never previously described, allows us to efficiently and correctly process the HSI and HSI-2a hybrid proteins, in which the SUMO sequence is connected by a peptide bond to the first amino acid residue of methionine-free interferon, connected to other amino acid residues of the protein by both the peptide and disulfide bonds . As a result, the enzymatic treatment of the HSI or HSI-2a fusion protein with ULP275 proteinase does not release the usual amino-terminal sequence, but the whole N-terminal structure of the methionine-free IFN-α2 containing a correctly closed disulfide bond.

This is the first discovered property of the SUMO system, consisting of the inventive SUMIN fusion protein and ULP275 proteinase, supplements the list of previously known properties of SUMO systems and expands the possibilities of their application.

The property of the SUMO system that we discovered is used in the claimed method by subjecting the HSI or HSI-2a fusion protein to enzyme treatment containing correctly established disulfide bonds, which allows enzymatic cleavage of the precursors of methionine-free IFN-α2 at the same time as their renaturation and leads to a significant reduction in the duration of the process of obtaining methomethine-free IFN-α2.

The enzyme proteinase ULP275 for the implementation of the proposed method is obtained using the engineered E. coli strain ECR-ULP275 (example 12). The biosynthesis, isolation and purification of ULP275 proteinase is carried out as described in example 17.

To implement the inventive method, E. coli strains are constructed: claimed - VKPM B-10414 and VKPM B-10700 and laboratory - Origami-HSI and Origami-HSI-2a - producers of the hybrid proteins HSI and HSI-2a - precursors of methionine-free interferons alpha-2b and alpha-2a, respectively, which, in addition to the SUMIN and SUMIN-2a fusion proteins, contain an additional N-terminal sequence of 10 histidine residues, which allows the proteins to be purified using metal chelate affinity chromatography on a Ni-NTA sorbent [Porath et al .., 1975, Nature 258: 598-599].

The process of obtaining the inventive strains-producers of hybrid proteins HSI and HSI-2a consists of several stages.

Step 1. Construction of recombinant plasmids

Recombinant plasmid DNAs are constructed based on the pET-28b (+) vector (Novagen), which provides inducible gene expression in E. coli cells under the control of the T7 RNA polymerase promoter. The constructed plasmids pET28-HSI and pET28-HSI-2a contain, in addition to the vector part, DNA fragments of 867 base pairs encoding the HSI or HSI-2a fusion proteins, the precursors of human methionine-free interferons alpha-2b or alpha-2a, respectively.

Stage 2. Transformation of recipient strains

As recipient strains, E. coli BL21 (DE3) and Origami 2 (DE3) (Novagen) strains using the T7 phage polymerase RNA gene on the chromosome are used. Plasmids pET28-HSI or pET28-HSI-2a are introduced into the cells of these strains using a transformation process. Cells of both strains are prepared for transformation by treatment with CaCl ₂ [Maniatis et al., 1984, M., Mir]. Transformed strains are selected on a selective medium containing the antibiotic kanamycin sulfate [Maniatis et al., 1984, M., Mir]. As a result of the transformation, E. coli strains are obtained: claimed, constructed on the basis of the recipient strain BL21 (DE3), and laboratory ones based on Origami 2 (DE3). The obtained strains in response to the introduction of an IPTG inducer into the culture medium [Maniatis et al., 1984, M., Mir] are able to synthesize hybrid proteins - the precursors of non-methionine interferons alpha-2b or alpha-2a person.

The inventive strains were deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as a strain of Escherichia coli VKPM B-10414 - producer of the precursor of methionine-free interferon alpha-2b (HSI fusion protein) and strain Escherichia coli VKPM B-10700 - producer of the precursor of bezmetionin-interferon-alpha-interferon-interferon-2 interferon (HSI-2a fusion protein).

The properties of the claimed strains VKPM B-10414 and VKPM B-10700

Morphological

Gram-negative, slightly mobile sticks with rounded ends 1.5-2.0 μm in length.

After 12 hours of growth at a temperature of 37 ° C on agar Luria [Maniatis et al., 1984, M., Mir] form whitish translucent colonies with a diameter of 1.5-2.5 mm; the surface of the colonies is smooth, the edges are even, the structure is homogeneous, the consistency is pasty, easily suspended in water.

After 40-48 hours of growth at a temperature of 37 ° C on M9 medium [Maniatis et al., 1984, M., Mir] form a grayish-whitish translucent, slightly convex with a shiny surface colony with a diameter of 0.5-1.5 mm.

Physiological and biochemical

Aerobes, gelatin are not diluted, indole is not formed.

As a carbon source, glucose, sucrose and various organic acids are absorbed, in some cases alcohols: ethanol, glycerin.

As a source of nitrogen, both mineral salts in ammonium and nitrate forms and organic compounds in the form of amino acids, peptone, tryptone, yeast extract, etc. are used.

As mineral salts, potassium phosphates, magnesium sulfate, sodium chloride, iron and manganese sulfates, chalk are used.

Resistant to antibiotic kanamycin at a concentration of up to 100 μg / ml.

Grow at a temperature of 20-40 ° C pH5.0 -9.0.

The inventive strains VKPM B-10414 and VKPM B-10700 and laboratory Origami-HSI and Origami-HSI-2a in response to induction using IPTG or lactose synthesize hybrid proteins HSI or HSI-2a - precursors of methionine-free interferons alpha-2b or alpha-2a human, respectively.

Storage conditions

In a lyophilized state at a temperature of -70 ° C.

For the implementation of the method, bacterial strains of E. coli are used that carry DNA fragments in the plasmid DNA encoding the synthesis of the HSI or HSI-2a fusion protein, the precursor of human methionine-free interferon alpha-2b or alpha-2a, respectively.

The strains are cultured in a suitable nutrient medium, including sources of carbon, nitrogen and mineral salts, usually used to grow E. coli cells at a temperature of 24 to 37 ° C [Maniatis et al., 1984, M., Mir]. Upon reaching the cultures of the strains of the early logarithmic growth phase, the IPTG inducer in a concentration of 0.1 to 2 mM or lactose in a concentration of 0.5 to 3% is introduced into the culture medium. After 0.5-10 hours of growth, the cells of producer strains accumulate the target HSI or HSI-2a proteins in the form of insoluble inclusion bodies in an amount of 5 to 30% of the total cell protein. Insoluble inclusion bodies are isolated and purified, and the synthesized HSI or HSI-2a fusion protein contained in them is subjected to dissolution [RU 2319502].

The renaturation of the HSI or HSI-2a protein is carried out as described [RU 2319502], except that before the renaturation is completed, the enzyme - ULP275 proteinase is introduced into the renaturation mixture. At this stage, three processes are carried out simultaneously in the renaturation mixture, namely, the end of renaturation and the end of the formation of disulfide bonds in the target HSI or HSI-2a proteins, as well as the enzymatic removal of SUMO from their composition under the action of the added LJLP275 proteinase. The obtained target product, renatured methionine-free interferon alpha-2b or human alpha-2a, containing correctly closed disulfide bonds, is subjected to final purification.

Purified non-methionine interferon alpha-2b or human alpha-2a has the following characteristics: a purity of at least 96%, the sequence of the first five N-terminal amino acids is identical to the sequence of native human non-methionine IFN-α2, specific activity is 2.0 × 10 ⁸ IU / mg.

Example 1. Cloning of the gene of human interferon alpha-2b

The human interferon alpha-2b gene is amplified in a PCR reaction using primers N466 (ataccatggaaaagagatgtgatctgcctcaaacccacagcctaggtagccgt) and N467 (atctcgagtcattctttacttcttatataaggg of template 50 of the X50 without changing the amino acid sequence of the encoded protein. A 510 bp DNA fragment obtained as a result of amplification elute from agarose gel using a Qiagen whale (Qiagen, cat. No. 28706), the eluted fragment is digested with restriction enzymes NcoI and XhoI and cloned in pUC18-NX vector containing the NcoI and XhoI sites in the modified polylinker of the pUC18 plasmid.

The result is the plasmid pUC18-IFN2b, in which the primary structure of the cloned human interferon alpha-2b gene is confirmed by determining its nucleotide sequence according to standard methods [Zimmermann J., Voss H., Schwager C., Stegemann J., Ansorge W. 1988 ; FEBS Lett. 233: 432-436] using standard pUC primers.

Plasmid pUC18-IFN2b carries the human interferon alpha-2b gene, the sequence of which contains a unique restriction enzyme site, XmaJ1.

Example 2. Cloning of the S. cerevisiae yeast SMT3 gene

The SMT3 gene is amplified by PCR using the S. cerevisiae Y618 strain as a chromosomal DNA template [Kartasheva et al., 1996, Yeast 12: 1297-1300]. Amplification is carried out in two stages. First, two overlapping DNA fragments are amplified, for which the following pairs of primers are used:

Fragment 1 with a size of 129 bp:

N450 (5'-atatccatggaaaagagatct gactcagaagtcaatcaagaa )

N454 (5'-cttgaagaaaatctctgaa)

Fragment 2, size 230 bp:

N453 (5'-ttcagagattttctttcaag)

N468 (5'-tacctaggctgtgggtttgaggcagatcacatccaccaatctgttctctgtga).

Amplified DNA fragments are eluted from an agarose gel, as in Example 1, and their mixture is used for PCR ligation. For this, PCR amplification is carried out on a mixture of fragments 1 and 2 as a matrix. The amplification primers are N450 and N468. The resulting 400 bp DNA fragment from PCR elute from agarose gel, digest with restriction enzymes NcoI and XmaJ1 and clone in plasmid pUC18-IFN2b (Example 1) containing the interferon alpha-2b gene and digested at the same sites. Cloning yields the plasmid pUC18-SUMO-IFN2b, in which the nucleotide sequence of the cloned SMT3 gene is verified by sequencing.

The resulting plasmid pUC18-SUMO-IFN2b contains a unique NcoI / XhoI DNA fragment encoding a hybrid protein gene, part of which is the SUMIN fusion protein (SEQ ID NO: 2), which is a human bezmethionine interferon alpha-2b sequence that is fused to the protein sequence SUMO yeast Saccharomyces cerevisiae.

Example 3. Construction of the HSI fusion protein gene

The SMT3 gene is amplified by PCR using the plasmid pUC18-SUMO-IFN2b as the DNA template. The amplification primers are N468 and N533 (5'-aaaagagatctcatcatcatcatcatcatcatcatcatcatggatccgactcagaagtcaatcaa). The amplified 366 bp DNA fragment elute from the agarose gel as in Example 1, digest with BglII and XmaJ1 restriction enzymes and ligate with the AatII / BglII fragment of the 484 bp pUC18-SUMO-IFN2b vector. and XmaJI / AatII fragment of the vector pUC18-SUMO-IFN2b with a size of 2652 bp (example 2). As a result of the manipulations, a sequence encoding the N-terminal extension of the protein, consisting of 10 histidine residues, is introduced into the 5'-terminal region of the SUMO structural gene. The result is the plasmid pUC18-HSI, which confirms the structure of the cloned DNA fragment.

Plasmid pUC18-HSI contains a unique NcoI / XhoI DNA fragment encoding the HSI fusion protein gene (SEQ ID NO: 1), the precursor of human bezmethionine interferon alpha-2b. An integral part of the HSI protein is the sequence of the SUMIN fusion protein (SEQ ID NO: 2).

Example 4. Cloning of the sequence encoding the proteinase ULP275

The DNA sequence encoding ULP275 proteinase is amplified by PCR using the laboratory strain S. cerevisiae Y618 as the chromosomal DNA template. The amplification primers are N447 (5'-atatggatcctctttggaaaggaaacataagga) and N448 (5'-attagaattctttaatgcatcggttaaaatcaaatgggcaat). The amplified DNA fragment with a size of 842 base pairs (bp) was eluted from an agarose gel, as in Example 1, treated with restriction enzymes BamHI and EcoRI and cloned into pUC18-His vector containing a polylinker DNA sequence flanked by EcoRI and XhoI sites and encoding a polypeptide consisting of 6 histidine residues.

The result is the plasmid pUC18-ULP275, which confirms the structure of the ULP275 gene by determining its nucleotide sequence by standard methods.

Plasmid pUC18-ULP275 contains a unique BamHI / XhoI DNA fragment encoding the ULP275 proteinase that carries a sequence of 6 histidine residues at the C-terminus that is responsible for the binding of the protein to the Ni-NTA sorbent under conditions of metal chelate chromatographic purification.

Example 5. Obtaining plasmids pET28-HSI

The recombinant plasmid pET28-HSI is a set of SalI / NcoI DNA fragment of the vector plasmid pET28b (+) (Novagen) size 5252 BP and the NcoI / XhoI DNA fragment of the plasmid pUC18-HSI (Example 3) with a size of 839 bp containing the HSI gene, a hybrid precursor of methionine-free interferon alpha 2b.

To obtain the plasmid pET28-HSI, the plasmid pUC18-HSI was digested with restriction enzymes NcoI and XhoI, an 839 bp DNA fragment was formed. elute from the gel and ligate with the DNA of the vector pET28b (+) digested with restriction enzymes NcoI and SalI. Ligation is carried out using T4 phage DNA ligase. The result is the plasmid pET28-HSI size 6091 bp

Plasmid pET28-HSI is expression; it is used for biosynthesis of the HSI fusion protein, which is necessary for the production of recombinant human methionine-free interferon alpha-2b. In the plasmid pET28-HSI, the HSI fusion protein gene is under the control of the T7 phage promoter.

Example 6. Obtaining plasmids pET28-HSI-2a

The human interferon alpha-2a gene is amplified by PCR using primers N467 (atctcgagtcattctttacttcttaaggattcttgcaagttt) and N617 (agcctaggtagccgtcgtaccttgatgctcctggcacagatgcgtaagatctctctcttt. The matrix is the plasmid DNA pSX50 [RU 2319502]. The use of such primers for amplification of a DNA fragment provides a change in the sequence of the human interferon alpha-2b gene to the sequence of the human interferon alpha-2a gene. The amplified DNA fragment size of 480 bp elute from agarose gel as in Example 1, digest with restriction enzymes XmaJI and XhoI and clone in the plasmid pET28-HSI (Example 5), cleaved at the same sites.

The result is the plasmid pET28-HSI-2a, in which, using sequencing, the correctness of the primary sequence of the cloned interferon alpha-2a gene is confirmed.

Plasmid pET28-HSI is expression; it is used for biosynthesis of the HSI-2a fusion protein, which is necessary for the production of recombinant human methionine-free interferon alpha-2a. An integral part of the HSI-2a protein is the sequence of the SUMIN-2a fusion protein (SEQ ID NO: 3), which is a sequence of human methionine-free interferon alpha-2a, which is fused to the SUMO yeast Saccharomyces cerevisiae protein sequence. In the plasmid pET28-HSI-2a, the HSI-2a fusion protein gene is under the control of the T7 phage promoter.

Example 7. Obtaining plasmids pET28-ULP275

Plasmid pET28-ULP275 is obtained on the basis of the vector pET28b (+) (Novagen) in several stages. First, a DNA fragment located between the NcoI and BamHI restriction sites is removed from the vector. For this, the pET28b (+) plasmid DNA is cleaved with restriction enzymes NcoI and BamHI, the sticky ends formed are blunt using the enzyme of the Klenow fragment of E. coli DNA polymerase I and ligated with T4 phage DNA ligase. As a result, plasmid pET28-NB1 is obtained, in the composition of which both used restriction sites are restored. The next step is to change the reading frame of the polypeptide initiated from the ATG codon, which is part of the NcoI site. For this, the plasmid pET28-NB1 DNA is digested with the restriction enzyme NcoI, the sticky ends formed are blunt using the enzyme of the Klenow fragment of the E. coli DNA polymerase I and ligated using the T4 phage DNA ligase. The result is the plasmid pET28-NB2, which is used to clone the proteinase gene ULP275.

To obtain plasmid pET28-ULP275, plasmid pUC18-ULP275 (Example 4) was digested with restriction enzymes BamHI and XhoI, resulting in a 855 bp DNA fragment. elute from the gel, as in Example 1, and ligate with the DNA of the vector pET28-NB2 digested with restriction enzymes BamHI and XhoI. The result is the plasmid pET28-ULP275 size 6091 bp

Plasmid pET28-ULP275 is used for biosynthesis of ULP275 proteinase. In the plasmid pET28-ULP275, the ULP275 proteinase gene is under the control of the T7 phage promoter.

Example 8. Construction of the claimed strain of E. coli VKPM B-10414

As the recipient strain, the strain E. coli BL21 (DE3) - VKPM B-6954 is used. Plasmid pET28-HSI (example 5) is introduced into the cells of the recipient strain using a transformation process using the CaCl ₂ reagent [Maniatis et al., 1984, M., Mir]. The transformant is selected on a selective medium containing the antibiotic kanamycin sulfate. As a result of the transformation, the claimed strain is obtained, which is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as Escherichia coli VKPM B-10414.

Strain VKPM B-10414 carries the expression plasmid pET28-HSI and, in response to introducing an IPTG inducer into the culture medium, is able to synthesize the HSI fusion protein, which is used to obtain methionine-free interferon alpha-2b.

Example 9. Construction of the claimed strain of E. coli VKPM B-10700

The strain VKPM B-10700 was constructed in the same way as the strain VKPM B-10414 (Example 8), except that the plasmid pET28-HSI-2a was used to transform the recipient strain E. coli BL21 (DE3) - VKPM B-6954 (example 6).

As a result of the transformation, the claimed strain is obtained, which is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as Escherichia coli VKPM B-10700.

Strain VKPM B-10700 carries the expression plasmid pET28-HSI-2a and, in response to introducing an IPTG inducer into the culture medium, is able to synthesize the HSI-2a fusion protein, which is used to produce non-methionine interferon alpha-2a.

Example 10. Construction of a laboratory strain of E. coli Origami-HS

To construct a laboratory E. coli strain, the E. coli Origami 2 (DE3) strain (Novagen), VKPM B-10187, carrying the T7 phage R7 polymerase gene on the chromosome, is used as the recipient strain. The construction of E. coli Origami-HSI strain is carried out as in example 8.

The result is a laboratory strain of E. coli Origami-HSI, which carries the expression plasmid pET28-HSI and is able to synthesize a hybrid HSI protein in response to introducing an IPTG inducer into the culture medium.

Example 11. Construction of a laboratory strain of E. coli Origami-HSI-2A

The laboratory strain of E. coli Origami-HSI-2a was constructed in the same way as the laboratory strain of E. coli Origami-HSI (Example 10), except that plasmid pET28-HSI-2a was used to transform the recipient strain (Example 6).

The result is a laboratory strain of E. coli Origami-HSI-2a, which carries the expression plasmid pET28-HSI-2a and is capable of synthesizing the HSI-2a fusion protein in response to introducing IPTG inducer into the culture medium.

Example 12. Construction of E. coli strain ECR-ULP275

The E. coli strain ECR-ULP275 was obtained by the method as in Example 8, except that the plasmid pET28-ULP275 (Example 7) was used to transform E. coli BL21 (DE3) - VKPM B-6954 recipient strain.

As a result of transformation, a strain of E. coli ECR-ULP275 is obtained. This strain carries the expression plasmid pET28-ULP275 and, in response to introducing an IPTG inducer into the culture medium, is able to synthesize ULP275 proteinase, which is used to obtain methionine-free interferon.

Example 13. Biosynthesis and isolation of the HSI hybrid protein from cells of the inventive strain VKPM B-10414

The strain is grown in flasks containing 50 ml of 2xYT medium (in wt.%): Tryptone - 1.6, yeast extract - 1, NaCl - 0.5, water - the rest, containing antibiotic kanamycin at a concentration of 40 μg / ml for 16-18 hours on an orbital rocking chair at a speed of 200-300 rpm at a temperature of 37 ° C. Then, 50 ml of fresh 2xYT medium containing the antibiotic kanamycin at a concentration of 40 μg / ml and an IPTG inducer at a concentration of 80 μM are added to the flasks. After this, cultivation is continued for 3 hours.

Cell biomass is separated from the medium by centrifugation (15,000 g).

From the obtained biomass, inclusion bodies containing an insoluble form of the HSI protein, the precursor of methionine-free interferon, are isolated and washed. Isolation and washing of inclusion bodies is carried out in the same way as the isolation and purification of an insoluble form of interferon [RU 2319502]. The result is a coarse fraction of insoluble inclusion bodies containing the precursor of methionine-free interferon with a yield of at least 10% of the wet weight of cell biomass.

Example 14. Biosynthesis and isolation of the HSI-2a fusion protein from cells of the inventive strain VKPM B-10700

The biosynthesis and isolation of the HSI-2a fusion protein is carried out in the same way as the HSI protein (Example 13), except that the inventive strain VKPM B-10700 serves as the producer strain. The result is a coarse fraction of insoluble inclusion bodies containing the precursor of methionine-free interferon with a yield of at least 10% of the wet weight of cell biomass.

Example 15. The biosynthesis of a hybrid HSI protein by cells of a laboratory strain of Origami-HSI

The biosynthesis of the HSI fusion protein by cells of the laboratory Origami-HSI strain is carried out as in Example 13, except that the Origami-HSI laboratory strain is the producer strain. Under induction conditions, cells of the laboratory Origami-HSI strain accumulate the target HSI protein in an amount of at least 10% of the total cell protein.

Example 16. The biosynthesis of the hybrid protein HSI-2a cells of the laboratory strain Origami-HSI-2a

The biosynthesis of the HSI-2a fusion protein by cells of the laboratory Origami-HSI-2a strain is carried out as in Example 13, except that the laboratory strain Origami-HSI-2a is the producer strain. Under conditions of induction, the cells of the laboratory strain Origami-HSI-2a accumulate the target protein HSI-2a in an amount of at least 10% of the total cell protein.

Example 17. The biosynthesis, isolation and purification of proteinase ULP275

2-3 individual colonies of transformed cells of the ECR-ULP275 strain are inoculated into test tubes in 5 ml of 2xYT medium containing the antibiotic kanamycin at a concentration of 40 μg / ml and glucose at a concentration of 20 g / l, and grown for 16-18 hours on an orbital shaker with speed of 200-300 rpm at a temperature of 37 ° C. Cells are harvested by centrifugation and suspended in 100 ml of fresh 2xYT medium containing kanamycin antibiotic at a concentration of 40 μg / ml. The cell suspension was transferred to flasks and cultured under the same conditions for 1 hour, after which an IPTG inducer was added to the growing culture to a concentration of 40 μM. Induction is carried out for 3 hours, culturing the cells under the same conditions.

Cells are harvested by centrifugation (15,000 g) and suspended in 5 ml of cell lysis buffer (LB buffer) of 50 mM Na _x H _{3 x} PO ₄ , pH 8.0; 300 mM NaCl; 20 mM Imidazole pH 8.0; water is the rest. Lysozyme is added to the cell suspension to a concentration of 1 mg / ml and the mixture is kept for 30 minutes in ice. The suspension is voiced in ice 6 times for 30 seconds with 0.5-1.0 'breaks for cooling at an ultrasonic device power of 200-300W. The lysate is clarified by centrifuging it for 30 minutes at 10,000 g at a temperature of 4 ° C.

A column containing 1 ml of Ni-NTA agarose (Qiagen) is washed with 5 ml of LB buffer, after which a clarified lysate is applied to the column at a flow rate of 1 ml / min. The column is washed successively with 10 ml of LB buffer, 10 ml of washing buffer with a composition of 50 mM Na _x H _3-x PO ₄ , pH 6.0; 300 mM NaCl; 10% glycerin; water - the rest, containing Imidazole pH 6.0 at a concentration of 20 mm; and 5 ml of wash buffer containing Imidazole pH 6.0 at a concentration of 150 mm. After this, the protein is eluted from the column using 2 ml of washing buffer containing Imidazole pH 6.0 at a concentration of 450 mM. Tween-20 is added to the resulting eluate to a concentration of 0.1%.

The resulting eluate was dialyzed against a buffer containing 50 mM Tris-Hcl pH 8.0, 150 mM NaCl, 2 mM dithiothreitol and 0.1% Tween-20.

The concentration of the eluted protein is measured spectrophotometrically at a wavelength of 280 nm. To calculate the protein concentration, the extinction coefficient Kex = 0.94 is used.

After measuring the concentration, the dialyzed preparation of ULP275 proteinase protein is diluted 2 times with glycerol and stored at -18 ° C.

Example 18. The preparation and purification of bezmethionine interferon alpha-2b person

The preparation and purification of human interferon alpha-2b without the N-terminal methionine using the HSI fusion protein is carried out in several stages.

Step 1. Dissolution, renaturation and processing of the HIS fusion protein

To the resulting suspension of inclusion bodies containing an insoluble form of the HSI protein (Example 13), dry guanidine hydrochloride (final concentration 6 M) or urea (final concentration 8 M), Tris-HCl solution, pH 8.0 (final concentration 20 mM) are added. ), EDTA (final concentration 5 mM), dithiothreitol (final concentration 50 mM). The resulting mixture was incubated at room temperature for 2 hours. The undissolved material is removed by centrifugation (15000 g), and the solution is subjected to sterilizing filtration through a membrane with a pore diameter of 0.22 μm.

HSI protein renaturation is carried out by diluting the resulting solution 100 times with a buffer of the composition: 20 mM Tris-Hcl (pH 8.0), 35 mM NaCl, 0.8 M urea, 0.1% Triton X-114, 1 mM oxidized glutathione, 1 mM reduced glutathione, water - the rest. Renaturation is carried out for 18 hours at a temperature of + 5 ÷ 12 ° C and constant stirring at a speed of 80 rpm

After this time, a solution containing ULP-275 proteinase at the rate of 4 mg proteinase per 12 g of inclusion bodies is added to the renaturation mixture with stirring at a speed of 120 rpm. The resulting mixture was incubated for 3 hours at a temperature of + 12 ° C with constant stirring at a speed of 80 rpm

Enzymatic cleavage of the HSI protein by ULP-275 proteinase is stopped by dilution and acidification of the reaction mixture. For this, the following components are added to the reaction mixture with stirring at a speed of 120 rpm in the indicated sequence:

- 0.25 M EDTA solution, pH 8.0 to a final concentration of 1 mm;

- purified water, cooled to a temperature of + 5 ° C, up to 25% of the volume of the renaturation mixture;

- 2.5 M sodium acetate solution, pH 4.5, to a final concentration of 12.5 mm.

The resulting mixture was filtered using a filter with a nominal pore size of 0.45 μm. The resulting solution of the target protein is called solution R.

Stage 2. Capture of the target protein

All subsequent stages of purification of non-methionine interferon alpha-2b person is carried out using an Akta Purifier (GE Healthcare) liquid chromatography system at a temperature of + 2 ÷ 8 ° C.

The target protein capture procedure was carried out using a HC 50/20 column (GE Healthcare) with a working volume of 250 ml containing Fast Flow carboxymethyl sepharose sorbent (GE Healthcare). Operating flow rate 30 ml / min. The column is equilibrated with 650 ml of B-8 buffer of 50 mM sodium acetate pH 5.0, 1 mM EDTA, water - the rest, after which a solution of the renatured and processed target protein is solution P. After the protein is applied, the column is washed with 4 L of B-8 buffer. The target protein is eluted with a simple linear gradient of 0-100% B-9 buffer of 50 mM sodium acetate, 0.5 M sodium chloride, 1 mM EDTA, pH 5.5, water - the rest, a gradient volume of 1200 ml. The elution of methionine-free interferon alpha-2b is monitored by optical density at a wavelength of 280 nm. Obtained at this stage, the solution of methionine-free interferon alpha-2b is called CM-1 solution.

Step 3. Reverse Phase Chromatography

The procedure is carried out using a column with a working volume of 400 ml containing sorbent C18. The working flow rate is 50 ml / min. The column is balanced with 700 ml of buffer A composition: 30% acetonitrile, 0.2% trifluoroacetic acid, water - the rest. After that, the solution CM-1 is applied to the column and the column is washed with 700 ml of buffer A.

Elution of non-methionine interferon alpha-2b is carried out with a complex linear gradient of 0-100% buffer. Composition: 80% acetonitrile, 0.2% trifluoroacetic acid, water - the rest. For elution use 3 l of buffer B. Elution is carried out sequentially according to the following scheme (buffer B in buffer A):

- 0-20% - 300 ml;

- 20-28% - 500 ml;

- 28-40% - 1.5 l;

- 40-60% - 600 ml;

- 60-100% - 100 ml

The elution of the methionine-free interferon alpha-2b is monitored by measuring the optical density of the eluate solution at a wavelength of 280 nm. Upon reaching 50 mUE, 8 ml fractions are collected in glass tubes containing 4 ml of B-11 buffer. After elution, the column is washed with 700 ml of buffer B.

The collected eluate fractions were analyzed by HPLC. Fractions containing methionine-free interferon alfa-2b with a purity of not less than 90% are combined and diluted 3 times with buffer B-11 of the composition: 12.5 mm sodium acetate, 1 mm EDTA, pH 4.5. The resulting solution is called - OF-1 solution.

Stage 4. Concentration

The procedure is carried out using a HC 50/20 column (GE Healthcare) with a working volume of 250 ml containing Fast Flow carboxymethyl sepharose sorbent (GE Healthcare). The working flow rate is 30 ml / min. The column was equilibrated with 650 ml of B-8 buffer, after which the OF-1 solution was applied and the column was washed with 650 ml of B-8 buffer. Elution is carried out at a flow rate of 15 ml / min with a simple linear gradient of 0-100% buffer B-9 with a length of 400 ml. The elution of the methionine-free interferon alpha-2b is monitored by measuring the optical density of the eluate solution at a wavelength of 280 nm. When a value of 50 mUE is reached, 10 ml fractions are collected in glass tubes. After elution, the column is washed with 350 ml of B-9 buffer.

The collected eluate fractions were analyzed by HPLC. Fractions containing the target protein with a purity of at least 96% are passed through a filter with a pore diameter of 0.22 μm, after which the resulting solution of methionine-free interferon alpha-2b is called - solution SM-2.

Step 5. Gel Filtration

The procedure is carried out using a column (GE Healthcare) with a working volume of 1700 ml containing Superdex-75 sorbent (GE Healthcare). The working flow rate is 5 ml / min. The column is balanced with 1.7 L of B-10 buffer of the composition: 25 mM sodium acetate, 150 mM sodium chloride, 0.5 mM EDTA, pH 5.1, water - the rest, after which a solution of SM-2 is applied and the column is washed with 1.7 L of buffer AT 10. The elution of the methionine-free interferon alpha-2b is monitored by measuring the optical density of the eluate solution at a wavelength of 280 nm. When a value of 30 mUE is reached, fractions are collected in glass tubes.

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

The result is a highly purified (with a purity of not less than 96%) preparation of human methionine-free interferon alpha-2b,

The authenticity, concentration and purity of the protein in the resulting preparation is analyzed by vertical polyacrylamide gel electrophoresis with / without the addition of DTT, with silver staining, as well as by HPLC [Eur. Pharm., 5 ^th ed .: 2004]. The described method allows to obtain at least 500 mg of methionine-free interferon alpha-2b from 10 g of the coarse fraction of inclusion bodies containing the HSI protein, the precursor of methionine-free interferon alpha-2b.

Example 19. The preparation and purification of bezmethionine interferon alpha-2a person

The preparation and purification of human interferon alpha-2a without an N-terminal methionine is carried out in the same manner as interferon alpha-2b, except that the HSI-2a fusion protein is used (Example 14).

The result is a highly purified (with a purity of not less than 96%) preparation of human interferon alpha-2a that does not contain N-terminal methionine.

The authenticity, concentration and purity of the protein in the resulting preparation is analyzed by vertical polyacrylamide gel electrophoresis with / without the addition of DTT, with silver staining, as well as by HPLC. The described method allows to obtain at least 500 mg of methionine-free interferon alpha-2a from 10 g of the coarse fraction of inclusion bodies containing the HSI-2a protein, the precursor of methionine-free interferon alpha-2a.

Example 20. Determination of the N-terminal sequence and the specific biological activity of interferon alpha-2b

The sequence of the five N-terminal amino acids of the isolated and purified interferon alpha-2b is determined (Example 18). The analysis is performed by the Edman method [Edman, 1950] on a Precise Sequencing System sequencer, model 492 (Applied Biosystems). The sequence determined by the analysis exactly coincides with the sequence of the first five amino acid residues of mature natural human interferon alpha-2.

The specific biological activity of isolated and purified interferon alpha-2b was determined by La Bormardiere & Laude [1981] in a culture of MDBK transplantable cell lines (ATCC No. CCL-22) sensitive to alpha-type interferon in comparison with the international standard sample (MCO) (WHO) International Standard INTERFERON ALPHA 2b, (Human rDNA derived) NIBSC code 95/566) against the indicator virus. As an indicator virus, use the virus of vesicular stomatitis (BBC) strain "Indiana" GKV GU Research Institute of Virology named. DI. Ivanovo RAMS (deposit No. 600) with an infectious titer of at least 10 ^-5 TCD ₅₀ / ml or the mouse encephalomyocarditis virus (EMC) STB GU Research Institute of Virology named after DI. Ivanovo RAMS (deposit No. 787) with an infectious titer of at least 10 ^-5 TCD ₅₀ in 1 ml.

The specific activity of the resulting interferon alpha-2b is 2.0 × 10 ⁸ IU / mg.

Thus, the claimed group of inventions allows:

- to simplify the process of obtaining biologically active non-methionine IFN-α2, through the use of an effective system of enzymatic removal of N-terminal methionine;

- reduce labor costs, time and expense of reagents for its production, due to the lack of the need for separate stages of the renaturation of IFN-α2, the formation of disulfide bonds in it and the enzymatic removal of the N-terminal methionine;

- to facilitate the solution of the task of purification of native IFN-α2 from other reaction products, due to the use of the highly efficient and specific proteinase ULP275 in the process of enzymatic cleavage, as well as due to a significant difference in the physicochemical properties of methionine-free IFN-α2 and its hybrid precursor.

Attached to the claimed group of inventions are: descriptions of the engineered strain ECR-ULP275 producing ULP275 proteinase, conditions for its cultivation, as well as methods for isolating and purifying the synthesized proteinase.

Claims (6)

1. A hybrid protein is a precursor to bezmethionine interferon alpha-2, an integral part of which is the sequence of bezmethionine interferon alpha-2, fused to the sequence of SUMO yeast Saccharomyces cerevisiae protein, and containing an N-terminal sequence of 10 histidine residues. 2. The hybrid protein of claim 1, wherein the human non-methionine interferon alpha-2 sequence is an interferon alpha-2a sequence. 3. The hybrid protein of claim 1, wherein the human non-methionine interferon alpha-2 sequence is an interferon alpha-2b sequence. 4. The bacterial strain Escherichia coli VKPM B-10414 - producer of the hybrid protein according to claim 1 of the precursor of methionine-free interferon alpha-2b person. 5. The bacterial strain Escherichia coli VKPM B-10700 - producer of the hybrid protein according to claim 1 of the precursor of methionine-free interferon alpha-2a person. 6. A method of producing a methionine-free interferon alpha-2 human by microbiological synthesis and isolation of the protein - the precursor of methionine-free interferon alpha-2 human, followed by enzymatic cleavage of its composition methionine-free interferon, characterized in that the bacterial strain Escherichia coli is used as the producer of the protein precursor the producer of the hybrid protein is the precursor of methionine-free interferon alpha-2 according to claim 4 or 5, and for the enzymatic cleavage of methionine-free interferon called ULP275 proteinase.