RU2617943C2 - Recombinant dna fragment, encoding human alpha-fetoprotein (afp), containing "slow" leucine-encoding codons, expression plasmid, containing said fragment, saccharomyces cerevisiae cell, transformed with said plasmid, and method for obtaining human afp - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of biotechnology, can be used for obtaining human alpha-fetoprotein (AFP) with application of strains of Saccharomyces cerevisiae yeasts, containing recombinant AFP gene. Obtained are: recombinant human AFT, containing so-called "slow" codons, encoding leucine in conservative AFP sections, recombinant plasmid and Saccharomyces cerevisiae cell for expression and secretion of said recombinant AFP respectively.

EFFECT: invention makes it possible to increase production of active AFP.

9 cl, 16 dwg, 3 tbl, 6 ex

Description

Technical field

The present invention relates to genetic engineering, biotechnology, the microbiological and medical industries, in particular to a method for producing human alpha-fetoprotein (AFP) using Saccharomyces cerevisiae yeast strains containing a recombinant AFP gene containing the so-called "slow" (rare) codons encoding leucine.

Description of the Related Art

Alpha-fetoprotein (AFP) is the main component of mammalian embryonic blood serum, which is synthesized by the visceral endoderm of the yolk sac and cells of the embryonic liver. Immediately after birth, the level of AFP in serum decreases sharply, and its expression is practically not determined in healthy adults (Deutsch, H.F. (1991) Adv. Canc. Res. 56, 253-312). AFP synthesis resumes when liver tumors and germinogenic teratoblasts occur and, to a lesser extent, with chemical and mechanical damage to the liver, accompanied by regeneration, for example, in acute viral hepatitis or cirrhosis (Mizejewsky GJ (2002) Expert Rev. Anticancer. Ther. 2: 89-115 )

Human AFP is a glycoprotein consisting of 590 amino acids and containing about 4% carbohydrate component (Morinaga, T., et al. (1983) Proc. Natl. Acad. Sci. USA 80, 4604-4608; Pucci, P. et al. , (1991) Biochemistry 30, 5061-5066). One of the main properties of AFP is non-covalent sorption of various low molecular weight chemicals, such as polyunsaturated fatty acids, steroid hormones, metals, retinoids, hydrophobic antibiotics and others (Aussel, C. & Masseyeff, R. (1994) Biochem. Biophys. Res. Commun. Commun 119: 1122-1127; Deutsch HF (1994) J. Tumor Marker Oncol. 9: 11-14.). In the early stages of embryonic development, AFP replaces albumin as a vehicle for fatty acids and other low molecular weight substances (Deutsch, H.F. (1991) Adv. Canc. Res. 56, 253-312).

The AFP molecule consists of three globular structural domains bonded by 15 interchain disulfide bonds, which significantly complicates the process of assembling the tertiary structure of the protein (Morinaga, T., et al. (1983) Proc. Natl. Acad. Sci. USA 80, 4604-4608; Pucci, P. et al. (1991) Biochemistry 30, 5061-5066). In addition, an important structural element of the AFP molecule is the carbohydrate component, which ensures the correct reception and functioning of the molecule (Deutsch, H.F. (1991) Adv. Canc. Res. 56, 253-312).

In addition to the polypeptide chain, consisting of 590 amino acid residues, the structure of the molecule of serum embryonic AFP or secreted by hepatocarcinoma cells includes one oligosaccharide group associated with N-type asparagine glycosylation (Yamashita K. et al. (1993) Cancer Res. 53: 2970- 2975). The structure of the AFP oligosaccharide chain is heterogeneous and depends on various factors: the stage of development of hepatocarcinoma or the stage of development of the embryo. Oligosaccharides affect the structural properties of the AFP molecule and are part of antigenic determinants and receptor-binding centers (Deutsch, H.F. (1991) Adv. Canc. Res. 56, 253-312). Unlike serum AFP, recombinant AFP expressed in bacterial cells is non-glycosylated, which is a characteristic difference between the product described by Murgita (US Patents 6,331,611; 6,627,440; 6,416,734), and therefore has structural and functional properties that distinguish it from serum analogue and also from recombinant AFP expressed in yeast systems. It is known that the expression of heterologous proteins in yeast involves glycosylation of the same amino acid residues as the serum analog, but the structure of the oligosaccharides themselves varies greatly in composition, length and branching of the chain, which also determines certain differences in the structural and functional properties of the corresponding proteins (Hard K. et al. (1989) FEBS Lett. 248: 111).

AFP has a number of functional properties, which are currently being intensively studied. The classical concept of AFP as an analogue of embryonic serum albumin is currently supplemented by data on the ability of AFP to regulate cell growth, development and programmed cell death (Mizejewsky G.J. (2002) Expert Rev. Anticancer. Ther. 2: 89-115). In particular, it has been shown that recombinant AFP, similar to the serum and culture counterpart, is able to inhibit the growth of estrogen-dependent tumor and normal tissues (Bennett, JA et al., (1997) Breast Cancer Res. Treat. 45, 169-179; Bennet , JA et al., (1998) Clinical Cancer Research, 4, 2877-2884). Recently, it has been found that AFS oncosuppressive activity is carried out by the mechanism of apoptosis activation, characterized by typical morphological changes, growth arrest, cytotoxicity and DNA fragmentation (Semenkova, LN (1997) Tumor Biol. 18, 261-274; Dudich EI, et al. ( 1998) Tumor Biol. 19, 30-40; Dudich EI et al. (1999) Eur. J. Biochem. 266: 1-13; Semenkova, L., et al. (2003) Eur. J. Biochem. 70: 4388-4399).

Earlier studies have shown the ability of AFPs to regulate the differentiation and activation of immune cells. In particular, AFP is able to suppress immune cells activated by allo or autoantigens and inhibit the expression of genes of a number of cytokines (Yamashita, K. et al. (1993) Cancer Res. 53, 2970-2975; US Patent No. 5,965,528). On the other hand, AFP has a pronounced stimulating effect on the growth of immature bone marrow cells, stem cells and embryonic cells (Dudich E.I., et al. (1998) Tumor Biol. 19, 30-40; US Patent No. 6,627,440).

These properties of AFP, as well as the increased selectivity of AFP uptake by cancer cells (in vivo) (Uriel J. et al. (1989) in Mizejewsky GI, Jakobson HI (eds): Biological Properties of Alpha-Fetoprotein. Boca Raton, CRC Press, vol 2: 103-117) are the basis for the use of AFP in medicine as a therapeutic drug in the treatment of autoimmune (US Patent No. 5,965,528) and oncological diseases (US Patent No. 6,416,734; Mizejewsky GJ (2002) Expert Rev. Anticancer. Ther. 2: 89-115). In addition, AFP traditionally acts as an oncoembryonic marker, which is used in the diagnosis of cancer and pathology of embryonic development (Deutsch, H.F. (1991) Adv. Canc. Res. 56, 253-312). However, the use of natural AFP as a medicine is technologically impossible due to a shortage of raw materials and restrictions on the use of animal products in the Pharmaceutical Industry. Previously published data on the expression and purification of recombinant AFP (rAFP) obtained by culturing various microorganisms (Yamamoto R., et al. (1990) LifeSciences, 46: 1679-1686; Nishi S., et al. (1988) J. Biochem 104: 968-972 US Patent No. 5,206,153; US Patent No. 6,331,611). Thus, intracellular production of human rAFP was obtained in Saccharomyces cerevisiae (Yamamoto R., et al. (1990) LifeSciences, 46: 1679-1686; US Patent No. 5,206,153) and Escherichiacoli (US Patent No. 6,331,611; Boismenu R., et al. (1997) Protein Expression and Purification. 10: 10-26). Recombinant AFP expressed in Escherichia coli has been shown to retain the immunoregulatory and oncosuppressive activity of the embryonic analogue (Boismenu R., et al. (1997) Protein Expression and Purification. 10: 10-26; Bennett, JA et al. (1997) Breast Cancer Res. Treat. 45, 169-179). The main disadvantage of these expression systems is the inability to secrete a heterologous protein and the extremely low level of its production. In addition, obtaining the target product from the biomass of recombinant producer strains required additional denaturation and renaturation procedures, which led to a significant decrease in the yield of the product and, as a result, significantly increased its cost. Also, in the case of using bacterial expression systems, the problem of contamination of the product with shell lipopolysaccharides with known endotoxic activity is also important.

The closest technical solution for this invention is a producer strain of human AFP described by the authors of the present invention in the following work: Dudich E. et al. (2012) Protein Expression and Purification. 84: 94-107. The article describes the creation of highly effective strains of Saccharomyces cerevisiae YBS247 + / AFP and YBS247 + / AFPmut - producers of recombinant human AFP in secreted form with native glycosylation type or in the form of a non-glycosylated mutant transformed with a plasmid containing human AFP optimized for all codons Saccharomyces cerevisiae. It was also shown that the introduction of additional chaperones - the Bip protein involved in the correct closure of S-S bonds and isomerase disulfide (PDI) significantly increase the yield of human AFP upon heterologous expression in Saccharomyces cerevisiae yeast. It was also shown that the non-glycosylated human AFP mutant showed lower tumor suppression activity compared to native AFP, but it also has a higher statistically significant ability to induce inhibition (invitro) of lymphocyte proliferation compared to glycosylated recombinant and native human AFP. It has also been shown that glycosylation of recombinant human AFP is necessary for its effective folding and secretion. Moreover, the presence of oligosaccharide is not essential for tumor suppression activity, but it is essential for its immunoregulatory activity, which indicates the involvement of various molecular mechanisms in these types of biological activity of human AFP.

However, there is currently a convincing need to develop new recombinant strains with the ability to increase the production of recombinant human AFP due to increased expression of a heterologous protein with intracellular assembly of the native tertiary structure and subsequent secretion of the target product into the culture fluid.

A brief description of the present invention

The aim of the present invention is to further improve the technology of expression and secretion systems of recombinant human AFP and to obtain new recombinant strains with the ability to increased production of recombinant human AFP.

This goal was achieved by creating a recombinant human AFP gene in which all codons are optimized for expression in Saccharomyces cerevisiae cells, with the exception of the so-called "slow" codons encoding leucine. The use of such a gene in Saccharomyces cerevisiae cells, the producers of human AFP, allowed us to increase the production level of this target protein both in the case of the non-glycosylated AFP variant and in the case of the glycosylated AFP variant. Thus, the present invention has been completed.

One aspect of the present invention is the provision of a recombinant DNA fragment encoding a protein having human alpha-fetoprotein (AFP) activity optimized for expression in Saccharomyces cerevisiae yeast cells, wherein said DNA fragment contains “slow” codons encoding L-amino acid residues of leucine, corresponding to the provisions of 13, 24, 52, 68, 72, 79, 86, 107, 119, 160, 161, 194, 199, 200, 239, 307, 311, 315, 332, 346, 352, 361, 362, 372, 382, 392, 399, 403, 408, 445, 450, 451, 465, 496, 534, 537, 549, 560, 588 in the native sequence of human AFP.

Another aspect of the present invention is the provision of the above DNA fragment that encodes AFP with a native glycosylation site corresponding to position 232 in the native AFP sequence of a person, representing the L-amino acid residue of asparagine, or without it.

Another aspect of the present invention is the provision of the above DNA fragment, which is characterized by the nucleotide sequence shown in the List of sequences under the number SEQ ID NO: 9 or 10.

Another aspect of the present invention is the provision of a recombinant plasmid for expression of recombinant human AFP in Saccharomyces cerevisiae cells, in the following sequence containing the GAL1 promoter, a pre-pro MFα secretion site, the described DNA fragment, yeast CYC1 gene transcription termination site, TuA gene Tu8 fragment, gene Tu8, gene ampicillin resistance, Tu8 fragment of the TuA gene, and yeast URA3 selection marker.

Another aspect of the present invention is the provision of the plasmid described above, which is the pTy8 / rhAFPmut_CU or pTy8 / rhAFP_CU plasmid of FIG. 8 and 10 respectively.

Another aspect of the present invention is the provision of a Saccharomyces cerevisiae cell transformed with the plasmid described above and capable of secreting recombinant human AFP into the culture fluid.

Another aspect of the present invention is the provision of the cell described above, wherein said cell is a cell of a strain of Saccharomyces cerevisiae YBS247 + / AFPmut_CU or a cell of a strain of Saccharomyces cerevisiae YBS247 + / AFP_CU.

Another aspect of the present invention is the provision of a method for producing recombinant human AFP comprising the steps of culturing the cells described above in a nutrient medium and isolating recombinant human AFP from the culture fluid.

Another aspect of the present invention is the provision of the above method, in which cells of the Saccharomyces cerevisiae strain YBS247 + / AFPmut_CU or cells of the Saccharomyces cerevisiae strain YBS247 + / AFP_CU are used.

Brief Description of the Figures

In FIG. Figure 1 shows the results of the analysis of the codons of the nucleotide sequence of the human AFP gene according to the frequency of occurrence of "fast" and "slow" codons for human cells. Gray bars correspond to codon frequencies below 20%.

In FIG. Figure 2 shows the results of the analysis of codons of the nucleotide sequence of the mouse mouse AFP gene. musculus in frequency of occurrence of "fast" and "slow" codons for mouse cells. Gray bars correspond to codon frequencies below 20%.

In FIG. Figure 3 shows the results of the analysis of the codons of the nucleotide sequence of the rat Rattusnorvegicus AFP gene by the frequency of occurrence of “fast” and “slow” codons for rat cells. Gray bars correspond to codon frequencies below 20%.

In FIG. 4 shows the results of the analysis of the amino acid sequences of the AFP protein of a human, mouse and rat. Amino acids represented by “slow” codons with a frequency of occurrence below 40% are indicated in the frame; the amino acids represented by “slow” codons with a frequency of occurrence below 20% are indicated by знаком.

In FIG. Figure 5 shows the results of the analysis of the nucleotide sequence of the human AFP gene (rhAFP0) containing codons that are optimal for its expression in Saccharomyces cerevisiae yeast cells by the frequency of occurrence of fast and slow codons for Saccharomyces cerevisiae yeast cells.

In FIG. Figure 6 shows the amino acid sequence of deglycosylated (N232Q substitution, underlined) human AFP (rbAFP0_CU) and the corresponding AFP gene nucleotide sequence containing the optimal (fast) codons for expression in Saccharomyces cerevisiae yeast cells with substitutions for the "slow" leucine codons. Changes made are highlighted in dark background.

In FIG. 7 shows the results of the analysis of the nucleotide sequence of the human AFP gene containing codons that are optimal for its expression in Saccharomyces cerevisiae yeast cells with substitutions for “slow” leucine codons, in terms of the frequency of occurrence of “fast” and “slow” codons for Saccharomyces cerevisiae yeast cells.

In FIG. 8 shows the structure of the expression plasmid pTy8 / rhAFP0_CU. Legend: GAL1 - promoter of the GAL gene of yeast, MFalfa - pre-pro site of secretion of MFα, CYC1 - termination site of transcription of the gene CYC1 of yeast, Tu8 - Tu8 fragment of TuA gene, AP ^R - gene for resistance to ampicillin, URA3 - marker of selection of yeast URA3.

In FIG. Figure 9 shows the strategy of site-directed mutagenesis to restore the glycosylation site Q → N at position 232. The recombinant deglycosylated AFP gene (rhAFP0_CU) was used as a matrix.

In FIG. 10 shows the structure of the expression plasmid pTy8 / rhAFP_CU. The legend is the same as in FIG. 8.

In FIG. 11 shows the structure of the plasmid Yepl3.

In FIG. 12 shows electrophoresis under denaturing conditions of Saccharomyces cerevisiae yeast culture fluid. To (control) - strain YBS247 + / AFPmut containing the gene for deglycosylated AFP with all optimized codons; 1-9 - YBS247 + / AFPmut_CU transforming strains containing the deglycosylated AFP gene with "slow" codons encoding leucine, M - fermentas molecular weight markers # SM0671, the arrow indicates the molecular mass of AFP.

In FIG. Figure 13 shows electrophoresis under denaturing conditions of the Saccharomyces cerevisiae yeast culture fluid (K (control) - YBS247 + / AFP strain containing the glycosylated AFP gene with all optimized codons; 1-12 - YBS247 + / AFP CU transforming strains containing the slow glycosylated AFP gene with " "codons encoding leucine, M-markers of molecular weight Fermentas # SM0671, the arrow indicates the molecular weight of the AFP.

In FIG. 14 shows electrophoresis under denaturing conditions of various samples of human AFP. Marker proteins (94, 67, 43, 30, 20 kD). 1 - embryonic AFP (eAFP); 2 - glycosylated recombinant human AFP (rhAFP) isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFP; 3 - glycosylated recombinant human AFP (rhAFP_CU), isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFP_CU; 4 - deglycolized recombinant human AFP (rhAFPO) isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFPmut; 5 - deglycolized recombinant human AFP (rhАFР0_CU), isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFPmut_CU.

In FIG. Figure 15 shows the effect of various AFP samples on concanavalin A-induced proliferation of mouse spleen T cells. T cells were incubated with embryonic AFP (eAFP); glycosylated recombinant human AFP (rhAFP) isolated from the culture fluid of producer Saccharomyces cerevisiae YBS247 + / AFP; glycosylated recombinant human AFP (rhAFPCU) isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFPCU; deglycosylated recombinant human AFP (rhAFPO) isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFPmut; and deglycosylated recombinant human AFP (rhAFPOCU) isolated from the culture fluid of the producer Saccharomyces24uCUpU + UFU + YF. The concentration of each AFP sample in the culture medium corresponded to 300 μg / ml, cell proliferation was determined by the incorporation of [ ³ H] -thymidine. The data are presented as the mean ± standard deviation of one of the experiments.

In FIG. Figure 16 shows the effect of various rhAPP samples on the proliferation of tumor and normal cells. Raji and NIH3T3 cells were incubated with glycosylated recombinant human AFP (rhAFP) isolated from Saccharomyces cerevisiae YBS247 + / AFP culture fluid; glycosylated recombinant human AFP (rhAFP CU) isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFP_CU; deglycosylated recombinant human AFP (rhAFPO) isolated from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFPmut; and deglycosylated recombinant human AFP (rhAFPOCU) isolated from the culture fluid of the producer Saccharomyces24uCUpU + UFU + YF. The concentration of each of the rhAPP samples in the culture medium corresponded to 300 μg / ml. Cells were incubated with rhFAP for 48 hours, cell proliferative activity was determined using the commercial CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega) kit. The data are presented as the mean ± standard deviation of one of the experiments.

Detailed description of the present invention

It is known that in most organisms, the efficiency of protein synthesis correlates well with the frequencies of codons in the open reading frame. This phenomenon is considered to be the result of the accumulation in the genes encoding efficiently synthesized proteins of the most rapidly “readable” codons. The codon reading rate is associated with the concentration of the corresponding tRNA fractions. As a result of the analysis of dynamic models describing the process of mRNA translation with this hypothesis in mind, scientists have identified a number of patterns of codon composition of genes that should be formed during evolution. One of the consequences was the conclusion about the regularity of the separation of codons by the speed of reading them by the ribosome and the increase in the proportion of “fast” codons in the genes as their expression efficiency increases (James F Kane. Current Opinion in Biotechnology 1995, 6: 494-500).

Despite this, the authors of the present invention suggested that in the case of heterologous expression of proteins with a complex structure, the reconstruction of the codon profile of the wild-type gene nucleotide sequence can be justified. To this end, the authors of the present invention analyzed the nucleotide sequences of the native human AFP gene (SEQ ID NO: 1), mouse (SEQ ID NO: 3) and rat (SEQ ID NO: 5) from the point of view of "optimality" of their composition (Fig . 1-3). The amino acid sequences of human, mouse, and rat AFPs are shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively.

Analysis of the data presented (Fig. 1-4) confirms the previously put forward hypothesis about the importance of "slow" codons, since the distribution profile of codons coincides in different organisms. Based on the study, the following patterns were identified:

- despite the low degree of homology of the presented sequences (for the mouse - 65.8%, for rats - 65.1%), the vast majority of amino acids represented by "slow" codons (with a frequency of occurrence of less than 25%) are leucine;

- almost all leucines are located in conserved regions of amino acid sequences (Fig. 4);

- Clusters are often found, consisting of several leucins located in a row at a slight distance from each other, encoded by "slow" codons (codon prevalence of 20-25%) [graphical codon usage analyzer, http://gcua.schoedl.de/ ]. In particular, leucines in positions 68-72; 194-199-200; 307-311-315; 399-403-408; 450-451 with a frequency of occurrence of less than 20% and 160-161; 361-362, 534-537 with a frequency of occurrence of less than 25-30%;

- the arrangement of "slow" codons in 90% of cases is duplicated in different organisms.

From literature data it is known that the AFP protein has a rather complex structure - domain organization, the presence of multiple cysteine bonds, which determines the complex folding of this protein. In an article by Dudich E. et al. (2012) Protein Expression and Purification. 84: 94-107, it was shown that the introduction of additional chaperones, the Bip protein involved in the correct closure of S-S bonds and disulfide isomerase (PDI), significantly increases the yield of human AFP upon heterologous expression in Saccharomyces cerevisiae yeast. Thus, the presence of amino acid clusters containing rare codons is apparently necessary to slow down the translation rate of mRNA encoding the AFP protein in various organisms.

To confirm this assumption, the previously synthesized gene of deglycosylated (Ν232Q replacement) ΑΦΠ human rhAFP0 (Dudich E. et al. (2012) Protein Expression and Purification. 84: 94-107; GenBank KP966106; SEQ ID NO: 7), containing optimal for yeast Saccharomyces cerevisiae "fast" codons (Fig. 5), the following replacements were made for "fast" codons with "slow" codons encoding leucine at locations corresponding to leucine at positions 13, 24, 52, 68, 72, 79, 86, 107 , 119, 160, 161, 194, 199, 200, 239, 307, 311, 315, 332, 346, 352, 361, 362, 372, 382, 392, 399, 403, 408, 445, 450, 451, 465 , 496, 534, 537, 549, 560, 588 AFP person. The nucleotide sequence of the obtained human deglycosylated AFP gene (rhАfР0_CU), optimized for expression in Saccharomyces cerevisiae yeast but containing “slow” codons encoding leucine at the above positions, is shown in SEQ ID NO: 9 and FIG. 6. The results of the analysis of the nucleotide sequence of the obtained gene from the point of view of "optimality" of its composition for expression in Saccharomyces cerevisiae yeast cells are shown in FIG. 7.

Also, in order to increase the production of recombinant glycosylated AFP (rhAFP CU) in the previously obtained gene (SEQ ID NO: 9), which contains codons that are optimal for expression in Saccharomyces cerevisiae yeast cells, replacing the “slow” leucine codons, Q was replaced by N in 232 position to return the native glycosylation site. The site-directed mutagenesis strategy for repairing the Q → N glycosylation site at position 232 AFP of a person is shown in FIG. 9. The gene used is the recombinant deglycosylated AFP gene (rhAFP0_CU). The nucleotide sequence of the obtained human glycosylated AFP gene, optimized for expression in Saccharomyces cerevisiae yeast but containing "slow" codons encoding leucine, is shown in SEQ ID NO: 10.

In the framework of the present invention, human AFP as a glycosylated variant, the amino acid sequence of which is given in SEQ ID NO: 2, but a deglycosylated variant, the amino acid sequence of which is shown in SEQ ID NO: 8, may include deletion, substitution, insertion or addition of one or more amino acids to one or in many provisions, provided that the AFP activity of a person is not impaired. The term "human AFP activity" means the activity of a protein by non-covalent sorption of various low molecular weight chemicals, such as polyunsaturated fatty acids, steroid hormones, metals, retinoids, hydrophobic antibiotics and others. It is also known that natural AFP and its recombinant analogues exhibit selective immunosuppressive properties. The indicated immunoregulatory activity of rhFAP samples isolated from the culture fluid can be measured using the classical lymphocyte blast transformation reaction (Wang G.L. et al. (2006) Acta Pharmacol Sin., 27 (4): 437-46 or see Example 5).

The number of "several" amino acids varies depending on the position or type of amino acid residues in the tertiary structure of the protein. This is due to the following reasons. Some amino acids have a high degree of homology with each other and therefore such changes do not greatly affect the tertiary structure of the protein. Therefore, the mutant variant of human AFP according to the present invention can be a protein with homology of at least 30-50%, preferably 50-70%, relative to the complete amino acid sequence of native human AFP having human AFP activity.

In the present invention, the “L-amino acid residue corresponding to any position indicated by a certain number means the amino acid residue corresponding to the position indicated by this number in the amino acid sequence of SEQ ID NO: 2.

To determine the L-amino acid residue corresponding to a position indicated by a certain number, it is necessary to align the amino acid sequence of the native human AFP and the amino acid sequence of the human AFP of interest.

Substitution, deletion, insertion or addition of one or more amino acid residues must be conservative mutations in order to maintain protein activity. Conservative substitutions are typical conservative mutations. Examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gin, His or Lys, replacing Asn with Glu, Gin, Lys, His or Asp, replacing Asp with Asn, Glu or Gin, replacing Cys with Ser or Ala, replacing Gin with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gin, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gin, Arg or Tyr, replacing Not with Leu, Met, Val or Phe, replacing Leu with Ile, Met, Val or Phe, replacing Lys with Asn, Glu, Gin, His or Arg, replacing Met with Ile, Leu, Val or Phe, replacing Phe with Trp, Tur, Met, Ile or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tur, replacing Tur with His, Phe or Trp, and replacing Val with Met, Ile, or Leu.

DNA encoding essentially the same protein as the human AFP described above is obtained, for example, by modifying the nucleotide sequence, for example, by the method of site-directed mutagenesis so that one or more amino acid residues at a particular site involve deletion, replacement, insertion or adding.

Calculation methods such as BLAST, FASTA, and ClustalW can be used to evaluate the homology of proteins or DNA.

BLAST (Basic Local Alignment Search Tool) - a heuristic search algorithm used by the blastp, blastn, blastx, megablast, tblastn and tblastx programs; these programs attribute values to indicators using the statistical methods of Karlin, Samuel, and Stephen F. Altschul ("Methods for assessing the statistical significance of molecular sequence features by using generals coring schemes". Proc. Natl. Acad. Sci. USA, 1990, 87: 2264-68; "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-7). FASTA method described by W.R. Pearson (Rapid and Sensitive Sequence Comparison with FASTP and FASTA, Methods in Enzymology, 1990, 183: 63-98). The Clustal W method is described by Thompson J.D., Higgins D.G. and Gibson T.J. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res. 1994, 22: 4673-4680).

The recombinant plasmid according to the present invention contains a DNA fragment encoding a recombinant human AFP or its variant, under the control of a promoter that functions in a eukaryotic cell, in particular in Saccharomyces cerevisiae yeast cells. As the recombinant plasmids according to the present invention, any plasmids having the ability to express the target protein in such cells can be used. Specific examples of recombinant plasmids according to the present invention are plasmids pTy8 / rhAFPmut_CU and pTy8 / rhAFP_CU. These plasmids contain the target gene with a pre-site of MFα secretion, a fragment of the bacterial plasmid pUC18c gene for resistance to ampicillin, a selective marker of yeast URA3, the terminator of transcription CYC1 and Tu8 fragment of the TuA gene of the yeast Saccharomyces cerevisiae, necessary for multiple integration of the target gene (about 30 copies) to the chromosome of yeast.

These recombinant plasmids were constructed by cloning a gene encoding recombinant human AFP into plasmid pTu8 (Dudich E. et al. (2012) Protein Expression and Purification. 84: 94-107). When these plasmids are introduced into the cell, a high level of transcription of the target genes is achieved.

Using the created plasmids, any eukaryotic cell can be transformed, in particular, the Saccharomyces cerevisiae yeast cell susceptible to such transformation by the indicated plasmids. Cell selection is not critical, as the methodology and methods of transformation are well known to those skilled in the art. Although the level of expression of the target protein may vary depending on the type of cell and culturing conditions of the obtained transformant, the fact of expression of the target protein will take place subject to successful transformation of the recipient cell.

The yeast cells of the present invention are yeast cells transformed with the DNA fragment described above and capable of producing and secreting recombinant human AFP into the culture fluid.

The phrase “yeast cells transformed with a DNA fragment” means that the desired DNA fragment has been introduced into the yeast cell using methods known to one skilled in the art. Transformation of a yeast cell with a DNA fragment leads to an increase in the expression of a DNA fragment encoding the recombinant human AFP according to the present invention. The presence of the MFα signal sequence results in secretion of the produced antibody into the culture fluid. Methods of transformation of yeast cells include all known methods, for example, a modified version of the procedure described for S. cerevisiae (Gietzand Schiestl, 1996).

The phrase "Saccharomyces cerevisiae yeast" means that said yeast is classified as Saccharomyces cerevisiae (S. cerevisiae) according to a classification known to one skilled in the art of microbiology. Examples of Saccharomyces cerevisiae yeast suitable for transformation with the plasmids of the present invention include, but are not limited to, Saccharomyces cerevisiae YBS247 + yeast (Dudich E. Et al. (2012) Protein Expression and Purification. 84: 94-107). In this strain, using the renewable marker URA3, the GAL80 and PRB1 genes were deleted and additional copies of the KAR2 and PDI1 genes encoding the Bip protein involved in the correct closure of SS bonds and disulfide isomerase (PDI) were introduced, which significantly increased the yield of human AFP upon heterologous expression in yeast Saccharomyces cerevisiae. Specific examples of cells transformed with plasmids according to the present invention are cells of the Saccharomyces cerevisiae strain YBS247 + / AFPmut_CU or the Saccharomyces cerevisiae strain YBS247 + / AFP_CU.

The method according to the present invention is a method for producing recombinant human AFP, comprising growing yeast in a nutrient medium and isolating the resulting recombinant human AFP from the culture fluid.

An example of yeast cells suitable for the production of recombinant human AFP according to the present invention are, but are not limited to, Saccharomyces cerevisiae yeast cells. Specific examples of cells suitable for the production of the recombinant human AFP according to the present invention are cells of the Saccharomyces cerevisiae strain YBS247 + / AFPmut_CU or the Saccharomyces cerevisiae strain YBS247 + / AFP_CU.

In the present invention, the cultivation of cells, the accumulation and purification of the recombinant human AFP according to the present invention from the culture fluid and other fluids can be carried out by a method similar to traditional fermentation methods, when a certain protein is produced using microorganisms.

The growth medium can be either synthetic or natural, provided that the medium contains a carbon source, a nitrogen source, minerals and, if necessary, a suitable amount of the nutrients that yeast needs to grow. Carbon sources include carbohydrates such as glucose and sucrose, and various organic acids. Depending on the method of assimilation, the indicated microorganism can use alcohol, including methanol, ethanol, glycerin. Various ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, and digested fermented microorganisms, can be used as a nitrogen source. As a source of minerals, potassium monophosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. As vitamins, thiamine, yeast extract and the like can be used.

The cultivation is preferably carried out under aerobic conditions, such as cultivation with stirring, shaking with aeration, at a temperature of from 20 to 37 ° C, preferably from 28 to 30 ° C. The pH of the medium is usually maintained in the range from 3 to 8, preferably in the range from 6 to 7.5. The pH of the medium can be adjusted using ammonia, calcium carbonate, various bases and buffers. Typically, growing for 2 to 5 days leads to the accumulation of the recombinant human AFP according to the present invention in the culture fluid.

After growth, solid components, such as cells, can be removed from the culture fluid by centrifugation or filtration using a membrane, and then the recombinant human AFP according to the present invention can be isolated and purified by precipitation with salts using sodium sulfate or ammonium sulfate, affinity chromatography, ion exchange chromatography, etc.

Examples

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

Example 1. Obtaining expression plasmids containing the genes of deglycosylated and glycosylated human AFP containing substitutions for the "slow" codons of leucine.

The synthetic deglycosylated human AFP gene (rhAFP0_CU) containing substitutions for the "slow" leucine codons flanked by the Ncol and Xhol restriction sites for subsequent integration into the expression plasmid was obtained by PCR using Dir primers (SEQ ID NO: 11) and Rev (SEQ ID NO: 12). The nucleotide sequence of the rhAFP0_CU gene was sequenced to exclude errors obtained during PCR.

The resulting PCR fragment was transferred at the NcoI-XhoI restriction sites to the plasmid pUC18 / GAL1-pp, the plasmid was named pUC18 / GAL1-pp / rhAFP0_CU. The nucleotide sequence of the rhAFP0_CU gene was sequenced to exclude errors obtained during PCR. To obtain an expression cassette, the AFP gene with leucine substitutions (rhAFP0_CU) under the control of the highly efficient GAL1 promoter and the secretion region of the MFal yeast at the HindIII - XhoI restriction sites was transferred onto the rTu8 vector similar to the synthetic rhAFP0 gene containing only optimal yeast codons (Dudich et al. 2012) Protein Expression and Purification. 84: 94-107).

The resulting pTy8 / rhAFP0_CU expression vector contains the target rhAFP0_CU gene, the bacterial plasmid pUC18 fragment, the URA3 selective marker, the CYC1 and Tu8 transcription terminator, the Saccharomyces cerevisiae yeast TuA gene fragment, which is necessary for the multiple integration of the target gene (about 30 copies) in chromosome. )

In order to increase the production of recombinant glycosylated AFP (rhAFP_CU) in the previously obtained gene containing the codons optimal for expression in S. cerevisiae yeast cells with “slow” codons encoding leucine, Q was replaced with N at position 232 to return the glycosylation site. The restoration of the glycosylation site (replacement of Q232 → N232) was carried out by PCR in 2 stages, as shown in FIG. 9.

The recombinant deglycosylated AFP gene (rhAFP0_CU) was used as a template at the 1st stage of PCR. The resulting PCR fragments were isolated, purified and used as templates in stage 2. The following primers were used for PCR: N1 (SEQ ID NO: 13), N2 (SEQ ID NO: 14), N3 (SEQ ID NO: 15), and N4 (SEQ ID NO: 16).

The result was a synthetic human AFP gene with “slow” codons encoding leucine at these positions and a glycosylation site (rhAFP_CU). The nucleotide sequence of the rhAFP-CU gene was sequenced to exclude errors obtained during PCR. The resulting gene (rhAFP_CU) was transferred at the NcoI - XhoI restriction sites to plasmid pUC18 / GAL1-pp, the plasmid was named pUC 18 / GAL1-pp / rhAFP_CU.

To obtain an expression cassette, the AFP gene with leucine codon substitutions (rhAFP_CU) under the control of the highly efficient GAL1 promoter and the secretion region of the yeast MFα1 at the HindIII - XhoI restriction sites was transferred to the rTu8 vector (from the laboratory collection) similar to the synthetic rhAFP gene containing only optimal yeast codons E. et al. (2012) Protein Expression and Purification. 84: 94-107).

The resulting pTy8 / rhAFP_CU expression vector contains the target rhAFP_CU gene, the pUC18 bacterial plasmid fragment, the URA3 selective marker, the CYC1 transcription terminator, and the Tu8 Saccharomyces cerevisiae yeast TuA gene fragment, which is necessary for the multiple integration of the target gene (about 30 copies) into the chromosome. )

Example 2. Obtaining strains of S. Cerevisiae - producers of deglycosylated and glycosylated human AFP.

The yeast strain S. cerevisiae YBS247 (MATa leu2 ur3), registered in the All-Russian Collection of Microorganisms (VKPM), with minor modifications was used as the recipient strain, with the use of the renewable marker URA3 the GAL80 and PRB1 genes were deleted and additional copies of the KAR2 and PDI genes were introduced encoding a Bip protein involved in the correct closure of SS bonds and disulfide isomerase (PDI), respectively (Dudich E. et al. (2012) Protein Expression and Purification. 84: 94-107).

The resulting recipient strain YBS247 + (MATa Δgal80 Δprbl KAR2 + PDI + leu2 ur3) containing 3-5 copies of the KAR2 and PDI1 genes was transformed with either the plasmid pTy8 / rhAFP0_CU or the plasmid pTy8 / endonuclease RNAlase and a KspAI fragment of the Yepl3 plasmid containing the S. cerevisiae yeast Leu2 gene (FIG. 11).

The transformants were selected on a minimal medium (SD) of the following composition (g / l): KH ₂ PO ₄ - 1.0, MgSO ₄ ⋅ 7H ₂ O - 0.5, CaCl ₂ - 0.1, NaCl - 0.1, (NH ₄ ) ₂ SO ₄ - 3.5, glucose - 20.0, YNB (Difco) - 1.7. The resulting clones (9 clones of YBS247 + / AFPmut_CU in the case of transformation with plasmid pTy8 / rhAFP0_CU and 12 clones of YBS247 + / AFPmC_CU in the case of transformation with plasmid pTy8 / rhAFP_CU) were grown in test tubes at 30 ° C on a shaker (250 rpm) for 72 rpm) the following composition: yeast extract - 1%, peptone - 2%, glucose - 1.0%, ethanol - 1.5%, 0.05 M sodium phosphate buffer (pH 7.0). As a positive control, the recombinant human AFP producer YBS247 + / AFPmut was used, which was previously created on the basis of the optimized non-glycosylated human AFP gene rhAFP0 (Dudich E. et al. (2012) Protein Expression and Purification. 84: 94-107). Analysis of yeast culture fluid and estimation of the amount of recombinant human AFP was performed by SDS electrophoresis and ELISA. The data obtained are presented in tables 1 and 2, as well as in FIG. 12 and 13.

For further work, the YBS247 + / AFPmut_CU-2 transforming strain was selected, which, when cultured in test tubes, showed the maximum production of deglycosylated AFP protein and its secretion into the yeast culture fluid when cultured in test tubes. Thus, the introduction of sections with “slow” leucine codons into the optimized AFP gene sequence made it possible to increase the yield of the target product and achieve a yield of 33.5 mg / L.

Also for further work, the YBS247 + / AFP_CU-7 strain was selected, which revealed the maximum production of the glycosylated AFP protein and its secretion into the yeast culture fluid when cultured in test tubes. Moreover, all transformates were more productive than the control strain. Thus, the introduction of sections with “slow” leucine codons into the optimized AFP gene sequence made it possible to increase the yield of the target product and achieve a yield of 50.1 mg / L.

Example 3. Determination of the yield of the target product (rhAFP) from the culture fluids of the producer strains of Saccharomyces cerevisiae YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU.

To analyze the yield of the target product (rhAFP), the procedure for isolating rhAFP from one liter of culture fluid samples was carried out according to the method described in the article by Dudich E. et al. (2012) Prot. Exp. Purif., 84: 94-107. Samples of the culture fluid were obtained by culturing strains of Saccharomyces cerevisiae: YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU, as described in Example 2. The process of isolation of rhAFP included three stages: in the first stage, protein isolation was carried out using cat CM-Sepharose sorbent (GEHealthcare, Germany); in the second stage, the DEAE-SepharoseFF anion-exchange sorbent (GEHealthcare, Germany) was used and gel chromatography was performed in the third stage using SephacrylS-200 HR sorbent (AmershamPharmacia, USA).

Stage 1

One liter of culture fluid was diluted with 3 liters of deionized water, adjusted to pH 5.2 by titration with 5 mMHCl and applied to a column (2.5 × 10 cm) with CM-SepharoseFF balanced with start buffer 1 (20 tM sodium acetate buffer, pH 5.2). The column was washed with start buffer and the desired product was washed off with the addition of 0.14 M NaCl.

Stage 2.

The target product obtained in the first stage by elution with CM-SepharoseFF was diluted with an equal volume of deionized water, the pH was adjusted to 7.5 by titration with MTrispH 8.0 solution 2 and applied to a column (2.5 × 10 cm) with DEAE-Sepharose FF equilibrated with buffer 2 (20 tM sodium phosphate buffer, pH 7.5). The column was washed with start buffer 2 to remove components not bound to the sorbent, and the target product containing rhAFP was eluted with start buffer 2 with the addition of 0.3 MNaCl

Stage 3.

The fractions containing rhAPP obtained in step 2 were concentrated in an Amicon cell (YM-30 membrane). Concentrated fractions (150 mg) were applied to a column (2.5 × 100 cm) containing SephacrylS-200 HR and balanced with 10 mM sodium phosphate buffer, pH 7.4. The target product containing rhAFP, which emerged as the main peak (mol. Weight. 70 kDa), was collected.

To determine the amount of protein extracted from the culture fluid of each of the analyzed Saccharomyces cerevisiae strains: YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU, the volume of the main peak obtained in stage 3 was measured and the protein concentration in this peak was determined.

Determination of protein concentration in solutions was carried out according to the Belford method (Bradford MM (1976) Anal Biochem; 72: 248-54), using a solution of embryonic AFP as a control, as well as spectrophotometrically at 278 nm, taking into account an extinction coefficient of Ε _1% , _{278 nm} = 0.53 (Dudich I. et al. (1999) Biochemistry; 38 (32): 10406-14.

The amount of rhAFP obtained from a liter of culture fluid of each of the analyzed strains is presented in Table 3. According to the results of the analysis, 21.6 mg of the target product rhAFP and 35 mg of the target product rhAFP_CU were obtained from one liter of the culture fluid of the producer strain S. cerevisiae YBS2A7 + / AFP was obtained from one liter of culture fluid of the producer strain S. cerevisiae YBS247 + / AFP_CU. From one liter of the culture fluid of the producer strain S. cerevisiae YBS247 + / AFPmut, 11.36 mg of the target product phAFP0 was obtained, 23.1 mg of the target product of phAFP_CU was obtained from one liter of the culture fluid of the strain S. cerevisiae YBS247 + / AFPmut_CU. Thus, when culturing strains producing S. cerevisiae YBS247 + / AFP_CU and S. cerevisiae YBS247 + / AFPmut_CU containing the “slow” leucine codon in the human AFP gene, the yield of the target product (rhAPP) from the culture fluid significantly exceeded the yield of the target product obtained from one liter of culture fluid of producer strains of S. cerevisiae YBS247 + / AFP and S. cerevisiae YBS247 + / AFPmut containing the optimized AFP gene.

Example 4. Electrophoretic characterization of rhFA samples isolated from culture fluids of Saccharomyces cerevisiae YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU strains.

Samples of rfAFP were obtained from the culture fluid of Saccharomyces cerevisiae strains: YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU as described in Example 3. Fetal AFP (eAFP) was isolated from the human navel al. (1999) Biochemistry; 38 (32): 10406-14). Serum was applied to DEAE-Sepharose Fast Flow (Pharmacia, 27 × 4 cm), equilibrated with start buffer (0.01 M Tris-Hcl, pH 7.5, 0.1 M NaCl). The components not bound to the sorbent were washed off with the start buffer, the target product was eluted with 0.2 M NaCl in the start buffer. The fractions containing AFPs were combined, the NaCl concentration was adjusted to 0.5 M and applied onto sequentially connected affinity columns with Sepharose CL-4B conjugated with rabbit polyclonal anti-AFP antibodies, rabbit polyclonal antibodies to human serum proteins and rat polyclonal antibodies against rabbit immunoglobulins equilibrated with 0.05 M Tris-HCl, pH 7.5 and 0.5 M NaCl. After the release of proteins that did not bind to antibodies, the adsorbed rfAPP was eluted with 0.005 M Hcl. The AFP solution was adjusted to pH 7.5 by the addition of a 2M Tris-HCl solution, pH 7.5. To obtain the monomeric form of AFP, an AFP solution obtained after affinity chromatography was applied to a column with Sephacryl S-200 (1.8 × 70 cm) in 0.01 M phosphate buffer, pH 7.0; 0.15 M NaCl. Natural AFP emerged as the main peak (mol. Weight. 70 kDa). For analysis of rhAPP samples, the Lammley gel electrophoresis method was used in 12.5% SDS-PAGE with β-mercaptoethanol followed by Coomassie staining. As seen in FIG. 14, natural embryonic AFP (eAFP), which was used as a standard, is represented by one protein band with a mol. weighing ≈ 69 kDa, corresponding to a fully glycosylated protein. Samples of glycosylated rhAFP isolated from the culture fluid of the producers Saccharomyces cerevisiae YBS247 + / AFP and Saccharomyces cerevisiae YBS247 + / AFP_CU - (rhAFP) and (rhAFP_CU), respectively, have a characteristic 2-strip profile with mol. weighing ≈ 70 kDa and 68 kDa. As noted earlier in the article by Dudich E. et al. (2012) Prot. Exp. Purif., 84: 94-107, the presence of two bands is a feature of rfAP produced by Saccharomyces cerevisiae YBS241 + / AFP, and is associated with the peculiarity of rhAPP glycosylation. A similar electrophoretic profile of rhAPP obtained from the culture fluid of the producer Saccharomyces cerevisiae YBS247 + / AFP_CU indicates the identity of AFP molecules. Samples of deglycosylated rhAFP obtained from the culture fluid of the producers Saccharomyces cerevisiae YBS247 + / AFPmut and YBS247 + / AFPmut_CU - (rhAFPO) and (rhAFP0_CU) respectively, also have the same electrophoretic profile and are characterized by the presence of one band with a mole. weighing ≈68 kDa. The presented data allow us to conclude that the rhFAP samples obtained from the culture fluid of yeast producers containing the optimized gene and the gene with leucine substitutions have an identical electrophoretic profile.

Example 5. Comparative analysis of the biological activity of samples of rhFAP isolated from the culture fluid (s) of the producer strains of Saccharomyces cerevisiae YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU.

Natural AFP and its recombinant analogs are known to exhibit selective immunosuppressive properties (Deutsch HF (1991) Adv. Cancer Res. 56, 253-312; Peck AB et al. (1978) J. Exp. Med. 148, 360-372, U.S. Patent No. 6,774,108). An analysis of the immunoregulatory activity of rhFAP samples isolated from the culture fluid of the Saccharomyces cerevisiae strains YB8247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU was performed using the classical lymphocyte (s) (Warmac) yeast (Blasttransforma) (b) lymphocyte transformation (s) (Warmac). : 437-46). Lymphocytes were isolated from the spleen of CBA mice recovered using sterile technique. To obtain a suspension of cells, the mouse spleen was passed through a nylon filter with a pore diameter of 40 μm. The red blood cells were removed by treating the Tris-NH ₄ Cl cell suspension with lysis buffer (0.144 mol / L NH ₄ Cl, 0.017 mol / L Tris-HCl). Cells were washed by centrifugation and placed in wells of a 96-well plate (Costar, USA) at 5 × 10 ⁵ cells per well in a volume of 200 μl. Cells were incubated in RPMI 1640 medium supplemented with 10% fetal calf serum (ECT), penicillin (100 U / ml), streptomycin (100 μg / ml) at 37 ° C in an atmosphere containing 5% CO ₂ in an incubator. Stimulation of lymphocyte proliferation was caused by the addition of 3 μg / ml ConA (concanavalin A). RhAFP samples were isolated from the culture fluid of Saccharomyces cerevisiae YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU strains, as described in Example 3. Natural AFP was isolated from cord blood serum Example 3 as described.

Samples of natural AFP and rhAFP were added to a suspension of ConA-activated lymphocytes and the cells were incubated for 72 hours. As a control, we used cells that were incubated without adding AFP samples. [ ³ H] -thymidine (1 μCi / well) was added to the reaction mixture in the last 18 hours. Cells were collected on glass filters using a Filtermat 196 cell harvester (Packard, USA). The ³ H-thymidine incorporation level was measured using a β-scintillation counter (BeckmanLS6500, USA).

In FIG. 15 presents the results of an analysis of the effect of AFP samples on the proliferative activity of ConA-activated mouse lymphocytes.

As can be seen from the data presented, all analyzed AFP samples at a concentration of 300 μg / ml cause significant inhibition of the incorporation of [ ³ H] -thymidine with ConA-activated mouse lymphocytes. The most pronounced ability to inhibit the proliferation of ConA-activated splenocytes was demonstrated by samples of deglycosylated rhAFP (rhAFP0 and rhAFP0_CU). It is characteristic that the specific activity of samples of deglycosylated rhAFP isolated from the culture fluid of the YBS247 + / AFPmut and YBS247 + / AFPmut_CU producers was almost identical, as the rhAFP0 sample caused 68% inhibition of cells, and the rhAFP0_CU sample induced 70% inhibition in the reaction. Samples of glycosylated rhAFP (rhAFP and rhAFP_CU) also inhibited the proliferative activity of ConA-activated splenocytes by 65 and 60%, respectively. Based on the presented data, it can be concluded that the rhAFP samples isolated from the culture fluid of yeast producers containing the optimized strain and strain with leucine substitutions exhibit identical biological activity and this activity corresponds to the activity of natural human AFP.

Example 6. Comparative analysis of the effect of rhFAP samples isolated from the culture fluid of the producer strains of Saccharomyces cerevisiae YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU on the proliferative activity of tumor and normal invitro cells.

The functional activity of various rhFAP samples was determined by their ability to influence the proliferative activity of Raji (cells of human B-cell lymphoma) and NIH3T3 (cells of mouse embryonic fibroblasts). Previously, the present inventors have shown that human rhFAP obtained from the culture fluid of the yeast producer Saccharomyces cerevisiae has the ability to inhibit the proliferation of tumor cells in humans and mammals and at the same time stimulates or does not affect normal inactivated human and mouse cells (Dudich EI et al. (1998) Tumor Biol. 1: 30-40; Dudich E. et al. (2012) Prot. Exp. Purif., 84: 94-107). The authors of the present invention used these invitro models to conduct a comparative analysis of the biological activity of various rhFAP samples.

NIH3T3 cells were cultured in DMEM supplemented with 10% 3CT (Sigma, USA) in a 5% CO ₂ atmosphere at 37 ° C. For reseeding, we used a solution of EDTA / 0.25% trypsin (PanEco, Russia). For the experiment, a cell suspension in DMEM containing 10% ECT was added to the wells of a 96-well plate (5 × 10 ³ cells / well in a volume of 100 μl) and placed in an incubator for 18-24 hours. On the day of the experiment, the medium was replaced with DMEM containing 2% ECT and the cells were placed in an incubator for 2 hours. Raji cells were cultured in RPMI medium supplemented with 10% ECT. Before the experiment, cells were washed in RPMI medium containing 2% ECT, introduced into the wells of a 96-well plate (1 × 10 ⁴ cells / well in a volume of 200 μl) and incubated for 2 hours. Then, 10 μl of solutions of different samples of rhAFP isolated from the culture fluid of the producer strains Saccharomyces cerevisiae YBS247 + / AFP, YBS247 + / AFP_CU, YBS247 + / AFPmut and YBS247 + / AFPmut_CU were added to the wells as described in Example R. Concentration corresponded to 300 μg / ml. Cells were incubated for 48 hours, control (wells) cells were incubated without the addition of rhFAP. To determine the proliferative activity of cells, a commercial Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay reagent was used (Promega, USA). The optical density of the reaction medium was measured at a wavelength of 490 nm using a Victor2 multifunctional counter (Wallack 1420, Finland).

As can be seen from FIG. 16, rhAPP samples isolated from the culture fluid (s) of producer strains containing the optimized gene and producer strains containing the optimized gene with "slow" leucine codons exhibit identical biological activity, inhibiting the proliferative activity of tumor cells. In experiments using a cell culture of mouse NIH3T3 fibroblasts (Fig. 16, NIH3T3 cells) it was also shown that rhAPP samples isolated from the culture fluid of producer strains containing the optimized gene and producer strains containing the optimized gene with "slow" non-leucine codons affect the proliferative activity of NIH3T3 cells.

Thus, it can be seen that samples of glycosylated rhAFP (rhAFP and rhAFP_CU) and deglycosylated rhAFP (rhAFP0 and rhAFP0_CU) isolated from the culture fluid of the producer strains Saccharomyces cerevisiae YBS247 + U AF24, UBS247U + MP AFP, YBS247 + P AF + AFP, YBS247 + P AF + AFP, YBS247 + P AF + AF AF, biological activity similar to that previously shown for natural (Dudich EI et al. (1998) Tumor Biol. 1: 30-40).

Although the present invention has been described in detail above, it will be apparent to those skilled in the art that changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention as defined by the appended claims.

Claims (9)

1. Recombinant DNA fragment encoding a protein having human alpha-fetoprotein (AFP) activity, optimized for expression in Saccharomyces cerevisiae yeast cells, characterized in that said DNA fragment contains “slow” codons encoding L-amino acid residues of leucine corresponding to the positions 13, 24, 52, 68, 72, 79, 86, 107, 119, 160, 161, 194, 199, 200, 239, 307, 311, 315, 332, 346, 352, 361, 362, 372, 382, 392, 399, 403, 408, 445, 450, 451, 465, 496, 534, 537, 549, 560, and 588 in the native human AFP sequence. 2. The recombinant DNA fragment according to claim 1, characterized in that said DNA fragment encodes AFP with a native glycosylation site corresponding to position 232 in the native human AFP sequence, which is an L-amino acid residue of asparagine, or without it. 3. The recombinant DNA fragment according to claim 1 or 2, characterized in that said DNA fragment is characterized by the nucleotide sequence shown in the Sequence Listing under the number SEQ ID NO: 9 or 10. 4. Recombinant plasmid for expression of recombinant human AFP in Saccharomyces cerevisiae cells, in the following sequence essentially consisting of the GAL1 promoter, a pre-pro site of secretion of MFα, a DNA fragment according to claim 1, a site of termination of transcription of the yeast CYC1 gene, TyA fragment of TyA gene, ampicillin resistance gene, Ty8 fragment of TyA gene, and yeast URA3 selection marker. 5. The recombinant plasmid according to claim 4, characterized in that said plasmid is plasmid pTy8 / rhAFPmut_CU or pTy8 / rhAFP_CU shown in FIG. 8 and 10 respectively. 6. A Saccharomyces cerevisiae cell transformed with the plasmid according to claim 4 and capable of secreting recombinant human AFP into the culture fluid. 7. The cell according to claim 6, characterized in that said cell is a cell of a strain of Saccharomyces cerevisiae YBS247 + / AFPmut_CU or a cell of a strain of Saccharomyces cerevisiae YBS247 + / AFP_CU. 8. A method for producing recombinant human AFP, comprising the steps of culturing cells according to claim 6 in a nutrient medium and isolating recombinant human AFP from the culture fluid. 9. The method according to p. 8, characterized in that the cells of the strain Saccharomyces cerevisiae YBS247 + / AFPmut_CU or the cells of the strain Saccharomyces cerevisiae YBS247 + / AFP_CU are used.

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