Microbiological synthesis method of target released protein in yeast saccharomyces cerevisiae

FIELD: biotechnologies.

SUBSTANCE: method involves cultivation in the appropriate conditions of yeast Saccharomyces cerevisiae and release of target protein; besides, release is directed with leader polypeptide, which has amino acid sequence SEQ ID NO1 and representing a variant of a pro-area of leader polypeptide of protein PpPIRl Pichia pastoris.

EFFECT: invention enlarges the range of methods for obtaining target protein in yeast Saccharomyces cerevisiae and increases possibilities for effective synthesis of such proteins.

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The invention relates to the field of biotechnology and relates to a method of synthesis of the desired secreted human protein in cells of the yeast Saccharomyces cerevisiae.

Under sekretiruemyi proteins we understand such targeted soluble proteins, biosynthesis which in yeast cells S. cerevisiae is accompanied by their secretion into the extracellular environment.

Obtaining proteins for technical and medical purposes using recombinant eukaryotic organisms is an important focus of the biotechnology industry. Significant place in the overall spectrum of products take secreted proteins, the production of which is more economical compared to intracellular, due to possibility of application of the simplified schemes of their isolation and purification. Among the microorganisms used for this purpose include the yeast S.cerevisiae.

It is known that the efficiency of secretion of proteins in yeast depends on the choice of directing the secretion leader polypeptides (leader regions) [Romanos et al., 1992, Yeast, 8: 423-488; Simonen et al., 1994, J Biol Chem, 269(19): 13887-13892; Murasugi, 2010, Recent Patents Biotechnology, 4: 153-166]. In the most General form of the leader polypeptide consists of the signal part (pre-region) and Pro-areas, each of them is removed from the composition of the secreted protein as perform their function.

The main functions of the leader polypeptide include:

- napravlenieah in the secretory pathways of the cell (pre - and Pro-regions);

- participation in the processes of folding and maturation of secreted proteins (mainly the function of the Pro-region) [Takagi & Takahashi, 2003, Appl Environ Biotechnol 63:1-9; Simonen et al., 1994, J Biol Chem, 269(19): 13887-13892];

- improving the solubility, i.e. thermodynamic stability of secreted proteins (Pro-region) [Kohara et al., 1994, Biosci Biotechnol Biochem, 58(4):779-81; Zhang et al., 2001, J Cell Biol. 153(6): 1187-98].

The method using the leader peptide (pre-Pro region) of α-factor in yeast S.cerevisiae, has established itself as an effective and versatile method for the synthesis of secreted proteins target [Romanos et al, 1992, Yeast 8, 423-488; Daly & Hearn, 2005, J. Mol. Recognit., 18: 119-138; Gellissen et al., 2005, FEMS Yeast Research 5: 1079-1096].

Other known secretory leader yeast origin is a pre-Pro-region of the glycoprotein HSP150 S. cerevisiae, a positive influence on the secretion of heterologous proteins in yeast cells installed, for example, β-lactamase [Simonen et al., 1994, J Biol Chem, 269: 13887-92] and the extracellular domain of the receptor for nerve growth factor in rats [Simonen et al., 1996, Yeast, 12: 457-66].

An example of a sequence of artificial origin, effectively performing in yeast cells the function of leader polypeptide is a fragment of the protein interleukin-1-beta [Lee et al., 1999, Biotechnol Prog, 15: 884-890; WO98/54339A1].

We have found several modifications of the Pro-regions leader of polypeptides that provide improved processing televi the proteins [Kjeldsen et al., 1996, Gene, 170: 107-112] and secretory activity [Kjeldsen, 2000, Appl Environ Biotechnol, 54: 277-286]. In particular, known artificially constructed Pro-peptide, providing a 4-fold increase in insulin secretion compared with the standard version of Pro-α-factor in yeast [Kjeldsen, 2000, Appl Environ Biotechnol, 54: 277-286].

However, none of the known leader of the polypeptides can be considered a versatile tool for the synthesis of secreted proteins in yeast cells.

For example, in respect of products of albumin in S.cerevisiae cells pre-Pro-region of α-factor mediates not increase secretion, and 5-6-fold increase in the degradation of this protein, whereas other leader polypeptides such effects do not detect [Sleep et al., 1990, Biotechnology, 8:42-46]. Destabilization of the target protein in the presence of α-factor leader has been observed in experiments on the expression of lysozyme silk worm cells P.pastoris [Koganesawa et al., 2001, Protein engineering, 14(9):705-710].

Different length fragments of the protein HSP1500 S. cerevisiae function as efficient secretory leader in the yeast S.cerevisiae, however, they have different transport possibilities and different opportunities to facilitate correct folding of secreted protein [Simonen et al., 1994, J Biol Chem, 269(19): 13887-13892; Simonen et al., 1996, Yeast, 12(5):457-66].

Also just one of the leaders in the Snov sequence of the protein HSP150 provides a high-level secretion of beta-lactamase in S.cerevisiae cells, while other variants leaders cause the accumulation of the protein in the cell or secretion of inactive form of the protein [Simonen et al., 1994, J Biol Chem, 269(19): 13887-13892].

In this regard, expanding Arsenal of leader polypeptide suitable for solving problems associated with the secretion of target proteins in yeast S.cerevisiae, is an important task.

As the closest analogue of the claimed invention, consider the way of microbiological synthesis of the desired secreted protein in the yeast S.cerevisiae using leader polypeptide comprising 45 amino acid residues of the subunit I protein HSP150 S.cerevisiae [WO93/18167].

Temperature-induced protein HSP150 the cell wall of the yeast S. cerevisiae has processed a leader polypeptide size 72 amino acid residue, of which the first 18 residues form a signal peptide, and the subsequent 54 residue forming subunit I protein function as a Pro-region [WO93/18167, Russo et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 3671-3675].

In addition to the capacity to use the top of the HSP150 protein fragments in cells of S. cerevisiae [Simonen et al., 1994, J Biol Chem, 269(19): 13887-13892; Simonen et al., 1996, Yeast, 12(5):457-66] know about the effective use of the leader region of the protein HSP150 S.cerevisiae as a secretory leader in yeast Pichia pastoris [Sievi et al., 2003, Biotechnol. Prog., 19: 1368-1371]. On the model of secretion of the catalytic domain of the α2,3-salitran feraz rats shows what the leader of the HSP150 able to send the correct folding and secretion of the protein in the cells of P. pastoris. The effectiveness of this leader region in the yeast P. pastoris and in yeast S.cerevisiae comparable with the efficiency of a universal α-factor leader [Sievi et al., 2003, Biotechnol. Prog 19: 1368-1371].

On the other hand known protein PpPIR1 yeast Pichia pastoris, homologous protein HSP150 S. cerevisiae and also have the leader polypeptide. It is known that the option of leader polypeptide protein PpPIR1 P.pastoris, have a size of 59 amino acid residues and homologous leader region of the protein HSP150 S.cerevisiae, can serve as a secretory leader in cells P.pastoris [Khasa et al., 2011, Yeast, 28(3):213-26].

Pro-region of the leader polypeptide protein HSP150 S. cerevisiae and Pro-region of the leader polypeptide protein PpPIR1 Pichia pastoris homologous to each other, however, contain a significant non-homologous areas and vary in length.

The objective of the claimed invention - expanding Arsenal of methods microbiological synthesis target of secreted proteins in the yeast Saccharomyces cerevisiae.

The problem is solved by developing a method of microbiological synthesis of the desired secreted protein by culturing in appropriate conditions, the yeast Saccharomyces cerevisiae, the secretion of the target protein in cells which directed the leader polypeptide, which includes a variant of the Pro-region of the leader polypeptide of the tree PpPIRl Pichia pastoris, the corresponding amino acid sequence of SEQ ID NO1.

Used in the present invention a variant of the Pro-region of the leader polypeptide protein PpPIR1 Pichia pastoris is different:

well - known variant of this Pro-region [Khasa et al., 2011, Yeast, 28(3):213-26] three amino acid substitutions, namely, at position 38 the amino acid residue is replaced by Asp Ser residue in position 39 the amino acid residue Lys replaced by a Met residue, and at position 40 the amino acid residue Il replaced by a Glu residue,

from HSP150 protein fragment of S. cerevisiae used in the method of the nearest similar, differs in size (42 residue in SEQ ID NO1 and no less than 45 residues in the nearest equivalent), and substitutions not less than 50% amino acid positions.

In the present invention used full option top of the polypeptide protein PpPIR1, which differs from the published version [Khasa et al., 2011, Yeast, 28(3):213-26] the above substitutions in the Pro region and the presence of additional methionine at the N - end.

The effectiveness of the proposed method is confirmed by the examples of synthesis in Saccharomyces cerevisiae cells serum human albumin and interferon Alfa-2b human

The method in General

For amplification of the fragment of the chromosomal DNA of the yeast P. pastoris, coding, leader polypeptide protein PpPIR1. using the polymerase chain reaction (PCR). So that is in the process of amplification change the sequence amplifierarava DNA fragment thus, to the modified sequence contains a unique website recognition of the restriction enzyme NcoI and code version of the modified polypeptide SEQ ID NO1, using additional primers. Amplificatory DNA fragment by means of genetic engineering is drained with a sequence of DNA having the promoter function in the yeast S. cerevisiae, and the resulting DNA fragment, precision fused with a DNA sequence that encodes a target secretory protein, clone in the expression vector. This expression vector can be epitomy or integrative vector capable of stably and selectively maintained in cells of the yeast S. cerevisiae.

Designed vector transformed cells of the recipient strain of the yeast S. cerevisiae. To do this, use one of the routine procedures of transformation. Selection of the transformants carried out by complementaly recessive chromosomal mutations recipient strain with a functional allele of the gene, localized in the composition of the vector DNA, or by use in the composition of the vector DNA of the gene encoding one of the dominant characteristics (for example, determining the resistance of cells to the action of antibiotics).

The obtained transformants used for the biosynthesis of secreted target protein. For this purpose, the cultivation of cells obtained what's transformants is carried out in a medium of the following composition, in wt.%: peptone 1-3; yeast extract 0.5 to 3; glucose 1-3, water - the rest, pH - natural within 16-48 hours at a temperature of between 22 and 32°C on a rocking chair 100-350 rpm

Under these conditions, yeast cells secrete into the environment of the cultivation of the target protein.

The method allows to synthesize different target secreted proteins, including serum albumin and interferon Alfa-2b human, in quantities that exceed or at least equal amounts of proteins synthesized using methods known from the previous level of technology.

The invention is illustrated by the following figures:

Fig 1. The influence of leader peptides on the secretion rate sivertone albumin person in the yeast S.cerevisiae. The secretion of serum albumin people directed leader following polypeptides:

HSA5 - signal peptide "ART" (strain HSA5-Sc),

HSA7 - leader of the polypeptide on the basis of the signal peptide "ART", merged with the Pro-region of the protein HSP150 yeast S.cerevisiae (strain HSA7-Sc),

HSA8 - full leader of the polypeptide protein PpPIR1 yeast P.pastoris, comprising the sequence SEQ ID NO1 (strain HSA8-Sc),

HSA9 - leader of the polypeptide on the basis of the signal peptide "ART", merged with the sequence SEQ ID NO1 Pro-region of the protein PpPIR1 yeast P.pastoris (strain HSA9-Sc).

Strains of S.cerevisiae HSA5-Sc and HSA7-Sc use of lead is turning polypeptides, known from the previous prior art, and considered as control strains HSA8-Sc and HSA9-Sc, secreting albumin person using the leader polypeptide comprising the sequence SEQ ID NO1, which is a variant of the Pro-region of the protein PpPIR1 P.pastoris. Comparative data on enzyme-linked immunosorbent assay (ELISA) samples of the culture fluid of the two experiments. Data is averaged over 3-10 random clones of transformants. Specified values of the standard deviation.

Figure 2. Electrophoregram samples of the culture liquid obtained by culturing yeast strains: IFN-F-Sc (path "α-factor), IFN-H-Sc (track PpPIR1"). Strain S.cerevisiae IFN-F-Sc uses the leader polypeptide, are known from the previous prior art, and considered as a control strain IFN-H-Sc, secreting interferon Alfa-2b human using leader polypeptide comprising the sequence SEQ ID NO1.

On the track caused the samples containing 5 µl of the culture liquid of each strain. The molecular weight markers (lane M, to the left shows the values of molecular weight protein markers (in kDa) served as proteins set PageRuler Prestained Protein Ladder (#SM0671, "Fermentas"). As a negative control (not able to synthesize interferon Alfa-2b people) caused the sample kulturalny fluid is recipientname strain S.cerevisiae G1L.

The invention is illustrated by the following examples.

Example 1. Cloning of a DNA fragment encoding the leader polypeptide protein PpPIR1 P.pastoris, including SEP ID NO1

Step 1. Amplification of genomic DNA fragment

The DNA fragment encoding the leader polypeptide protein PpPIR1 P.pastoris is obtained 2-stage PCR amplification of fragments of the chromosomal DNA of strain GS115 (Invitrogen, USA). Matrix amplification is chromosomal DNA of strain P.pastoris GS115, which emit method Sydoruk [Sidoruk et al., 2009]. On the 1st stage PCR amplification of fragment 1 using a pair of primers N520 (5'-attaaatgatcaaccatgatgtacaggaactt) and N541 (5'-tgtgctccaaggttcggaa), and fragment-2 using primers N540 (5'-ttccgaaccttggagcaca) and N521 ('-ttactcgagtctagatctcttttccatggaggcatctaaggacttaa). On the 2nd stage PCR amplification of fragment 3 using primers N520/N521 and mixtures of fragments 1 and 2 as a matrix.

By treatment with restriction endonucleases BclI and XhoI at the ends of the amplified fragment-3 DNA reveal the sticky ends of the respective restriction sites and thus get suitable for subsequent cloning the DNA fragment encoding the leader polypeptide protein PpPIR1 P.pastoris.

In the 2-stage amplification in a DNA sequence that encodes a leader polypeptide alter the unique location of website usnavi the Oia restrictase NcoI.

Step 2. Cloning of a DNA fragment encoding the leader polypeptide protein PpPIR1 P.pastoris comprising SEQ ID NO1

Plasmid pUC18x-GAL1-hsp150pp receive as a result of simultaneous ligation of three DNA fragments: 1) HindIII/XhoI fragment containing the vector part of the laboratory plasmids pUC18x; 2) HindIII/BamHI fragment containing the sequence of the promoter GAL1 yeast S.cerevisiae; 3) BclI/XhoI DNA fragment obtained in step 1 and encoding a leader polypeptide, protein PpPIR1 P.pastoris.

The resulting plasmid pUC18x-GAL1-hspl50pp consists HindIII/NcoI DNA fragment of the nucleotide sequence of the promoter GAL1 yeast S.cerevisiae, merged with the DNA sequence that encodes a leader polypeptide protein PpPIR1 P.pastoris.

Example 2. Construction of strains of yeast S.cerevisiae for the secretion of serum albumin human

Step 1. Design vector pgkU::HSA8

To this end receive three DNA fragments: 1) HindIII/SalI DNA fragment containing the vector part of the integrative vector R-3 [RU2420 567]; 2) Hindlll/Ncol fragment of DNA plasmids pUC18x-GAL1-hsp150pp, comprising the nucleotide sequence of the promoter GAL1 yeast S.cerevisiae, merged with the DNA sequence that encodes a leader polypeptide protein PpPIR1 P.pastoris; 3) NcoI/XhoI DNA fragment encoding the structural gene HSA serum albumin human, the resulting open end restriction enzymes cut sites NcoI XhoI fragment DNA which is amplified in a PCR reaction using primers N522 (5'-tatccatggaaaagagagacgctcacaagtcagaa) and N169 (5'-gagcggataacaatttcacacagg) and DNA vector R-63 (RU2009140367) as a matrix.

Vector pgkU::HSA8 receive as a result of simultaneous directional ligation of these three DNA fragments.

Vector pgkU::HSA8 designed for expression in yeast S.cerevisiae under control of the GAL1 promoter gene serum albumin human, the secretion of which it is a modified version of leader polypeptide protein PpPIR1 P.pastoris.

Step 2. Design vector pgU::HSA7

To this end HindIII/NcoI DNA fragment of the vector pgkU::HSA8, comprising the nucleotide sequence of the promoter GAL1 yeast S.cerevisiae, merged with the DNA sequence that encodes a leader polypeptide protein PpPIR1 P.pastoris, as opposed to HindIII/NcoI DNA fragment containing the nucleotide sequence of the promoter GAL1 yeast S.cerevisiae, merged with the DNA sequence that encodes a synthetic signal peptide "ART" (HindIII/ > PST fragment DNA vector R-35, RU2009140367), which in turn merged with the DNA sequence that encodes the Pro-region of the protein HSP150 yeast S.cerevisiae (>PST /NcoI DNA fragment of the vector R-16, RU2420567).

The result is a vector pgkU::HSA7, which is suitable for expression in yeast S.cerevisiae under control of the GAL1 promoter gene serum albumin human secretion is vtorogo aimed leader of the polypeptide on the basis of the signal peptide "ART", merged with the Pro-region of the protein HSP150 yeast S.cerevisiae.

Step 3. Design vector pgkU::HSA9

To this end > PST /NcoI DNA fragment of the vector pgkU::HSA7 containing the nucleotide sequence encoding the Pro-region of the protein HSP150 yeast S.cerevisiae, as opposed to > PST /NcoI DNA fragment encoding the Pro-region of the protein PpPIR1 P.pastoris. > PST /NcoI DNA fragment encoding the Pro-region of the protein PpPIR1 P.pastoris, get in the PCR amplification of the corresponding DNA fragment from plasmid pUC18x-GAL1-hsp150pp using primers

N542 (5'-tgcctgcagctttgggtgcctacgttccttccgaaccttggagcaca) and

N521 (5'-ttactcgagtctagatctcttttccatggaggcatctaaggacttaa) and the subsequent opening of the sticky ends of the amplified fragment by treatment with restriction endonucleases > PST and NcoI.

The result is a vector pgkU::HSA9 for gene expression of serum albumin person in S.cerevisiae cells under the control of the GALL promoter, the Secretion of this protein is aimed leader of the polypeptide on the basis of the signal peptide "ART", merged with the sequence SEQ ID NO1 Pro-region of the protein PpPIR1 P.pastoris.

Step 4. Construction of strains of yeast S.cerevisiae HSA5-Sc, HSA7-Sc, HSA8-Sc and HSA9-Sc

With this purpose, carry out the transformation of the recipient strain S. cerevisiae G1L, bulleted four auxotrophic mutations (RU2420567), vector R-35 (RU2009140367) or pgkU::HSA7 or pgkU::HSA8 or pgkU::HSA9, respectively.

Transformation recipientsa strain of yeast G1L Provo is drawn by the method of ito et al.(Ito N., Fukuda Y., Murata, K., Kimura, A. 1983. J Bacteriol., 153: 163-168). For selection of transformants using a synthetic environment with the addition of amino acids or bases, the corresponding auxotrophic mutations design strain to a final concentration of 30 μg/L. the synthetic environment, wt.%: KN2RHO4- 0,1, MgSO4to 0.05, NaCl And 0.01, CaCl2- 0,01, (NH4)2SO4- 0,35, glucose - 2, thiamin (vitamin B1) - 0,02, Riboflavin (vitamin B2) - 0,02, nicotinic acid (vitamin PP) - 0,02, p-aminobenzoic acid - 0,02, calcium Pantothenate - 0,02, Biotin - is 0.0002, pyridoxine (vitamin B6) - 0,02, Inositol - 1, folic acid and 0.02, water - the rest.

Immediately before the transformation of the DNA of each vector is treated with restriction enzyme XhoI, thereby removing from the integrable structure of the bacterial vector part and directing the integration of the vector in the locus PGK1 chromosomes of yeast.

As a result of transformation get 2 independent clone strains of yeast S.cerevisiae HSA5-Sc, HSA7-Sc, HSA8-Sc and HSA9-Sc, respectively. The secretion of serum albumin human cells derived strains of yeast are directed signal peptide "ART" (HSA5-Sc), leader of the polypeptide on the basis of the signal peptide "ART", merged with the Pro-region of Beja HSP150 yeast S.cerevisiae (HSA7-Sc), leader of the polypeptide on the basis of the signal peptide "ART", merged with the sequence SEQ ID NO1 Pro-region of the protein PpPIR yeast P.pastoris (HSA9-Sc) or full leader of the polypeptide protein PpPIR1 yeast P.pastoris, contains the sequence SEQ ID NO1 Pro-region (HSA8-Sc).

Strains of S.cerevisiae HSA5-Sc and HSA7-Sc are treated as control strains HSA8-Sc and HSA9-Sc, secreting albumin person using the leader polypeptide comprising the sequence SEQ ID NO1 Pro-region of the protein PpPIR1 P.pastoris.

Example 3. Construction of strains of yeast S.cerevisiae for the secretion of interferon Alfa-2b human

Step 1. Construction of recombinant plasmids pUC18x-IFN

With this purpose, carry out ligation of two DNA fragments: 1) a BamHI/XhoI fragment containing the vector part of the lab vector pUC18x; 2) BamHI/XhoI DNA fragment, which is obtained in the PCR amplification of the DNA fragment of the plasmid pUC18x-GAL1ppI-IFN2b (RU2427645) using primers N499 (5'-ataggatccteteatctecctcaaacccac) and N169 (5-gagcggataacaatttcacacagg) and the subsequent opening of his sticky ends using restricted BamHI and XhoI.

Plasmid pUC18x-IFN concludes the nucleotide sequence of the structural gene of interferon Alfa-2b men in BamHI/XhoI DNA fragment. Step 2. Design vector pPDX2H-sIFN

With this purpose, carry out simultaneous ligation of three DNA fragments: 1) HindIII/XhoI DNA fragment containing the vector part of the recombinant plasmids pPDX2-IFN2b (RU2427645); 2) HindIII/BgIII fragment of DNA plasmids pUC18x-GALl-hsp150pp, comprising the nucleotide sequence of the promoter GAL1 yeast S.cerevisiae, SL is the case with DNA sequence, the coding of the leader polypeptide protein PpPIR1 P.pastoris; 3) BamHI/XhoI DNA fragment of plasmid pUC18x-IFN containing the nucleotide sequence of the structural gene of interferon Alfa-2b human.

The result of replicative vector pPDX2H-sIFN, which is suitable for expression in yeast S.cerevisiae under control of the GAL1 promoter gene recombinant interferon Alfa-2b human, the secretion of which is aimed leading polypeptide protein PpPIR1 P.pastoris.

Step 3. Konstruirovanie vector pPDX2F-sIFN

To this end HindIII/NcoI fragment of DNA plasmids pPDX2H-sIFN replace with HindIII/NcoI fragment of DNA plasmids pUC18x-GAL1ppI-IFN2b [RU2427645], comprising the nucleotide sequence of the promoter GAL1 yeast S.cerevisiae, merged with the DNA sequence that encodes a leader polypeptide α-factor in yeast S.cerevisiae (pre-Pro-region of α-factor in yeast S.cerevisiae).

The result of replicative vector pPDX2F-sIFN, which is suitable for expression in yeast S.cerevisiae under control of the GAL1 promoter gene recombinant interferon Alfa-2b human, the secretion of which is aimed leading polypeptide α-factor of the yeast S.cerevisiae.

Step 4. Construction of strains of yeast S.cerevisiae IFN-H-Sc and IFN-F-Sc

With this purpose, carry out the transformation of the recipient strain S. cerevisiae G1L, bulleted four auxotrophic mutations (RU2420567), vector pPDX2H-sIFN or PDX2F-sIFN, respectively.

Transformation recipientsa strain of yeast G1L and selection of the transformants carried out as in example 2 except that the use of a circular vector DNA without exposing their cleavage.

As a result of transformation get 2 independent clone strains of yeast S.cerevisiae IFN-H-Sc and S.cerevisiae IFN-F-Sc, respectively. Secretion of recombinant interferon Alfa-2b human strain of S.cerevisiae IFN-H-Sc directed the leader polypeptide protein PpPIR1 P.pastoris (IFN-H-Sc), and the strain of S.cerevisiae IFN-F-Sc, which is the control, the leader polypeptide α-factor in yeast S.cerevisiae (IFN-F-Sc).

Example 4. The cultivation of the yeast S.cerevisiae and analysis of the level of secretion of the target protein

For growing strains of yeast S.cerevisiae IFN-H-Sc, IFN-F-Sc, HSA5-Sc, HSA7-Sc, HSA8-Sc and HSA9-Sc use complex YPD medium of the following composition, wt.%: peptone -2, yeast extract -1, -2 glucose, water - the rest. Yeast is cultivated at a temperature of +28°C on a shaker (250 rpm) for 42 hours at a density of seeding 5×105ml-1.

A comparative analysis of the level of secretion of interferon Alfa-2b human cell strains IFN-H-Sc and IFN-F-Sc is carried out by electrophoresis of proteins cultural liquid in 15% SDS page under denaturing reducing conditions using the Mini-PROTEAN Tetra Cell (#165-8000, "BioRad") according to the instructions that shipped with the system. Preparation of buffers for the preparation of samples of the culture fluid, drawing on the gel and electrophoresis carried out according to the instructions that shipped with the system. Use markers molecular weight PageRuler Prestained Protein Ladder (#SM0671, "Fermentas"). Staining the gel with silver nitrate carried out by means of a set PageSilver Silver Staining Kit "Fermentas" (#K0681, "Fermentas") according to the instructions attached to the kit.

A comparative analysis of the level of secretion of serum albumin person carried out using the method and the ELISA Protocol Albumin Human Bioassay ELISA Kit (A 1274-85, "US Biological"). As a negative control (not able to synthesize serum albumin) using the recipient strain of the yeast S.cerevisiae G1L.

The results of the comparative analysis (figure 1) show that the efficiency of synthesis in the yeast Saccharomyces cerevisiae secreted serum albumin person using leader polypeptides comprising variant of the Pro-region, SEQ ID NO1, exceeds (strain HSA8-Sc), or at least, not inferior (strain HSA9-SC) efficiency of synthesis of this protein with the use of leader polypeptide, are known from the previous level of technology (strains HSA5-Sc and HSA7-Sc).

The comparative analysis (figure 2) show that the efficiency of the synthesis of secreted interferon Alfa-2b human using leader polypeptide protein PpPIR1 P.pastoris, including option for Pro-region with amino acid sequence SEQ ID NO1 (strain IFN-HSc), not inferior to the efficiency of synthesis of this protein by using "universal" leader polypeptide α-factor of yeast, used as control (strain IFN-F-Sc). In addition, it is seen (figure 2)that the target product synthesized by the claimed method using strain IFN-H-Sc is of a higher quality than the target product synthesized by the control strain IFN-H-Sc, as it contains a much smaller number of background proteins.

Thus, a variant of the Pro-region of the leader polypeptide protein PpPIR1 Pichia pastoris, the corresponding amino acid sequence of SEQ ID NO1, which developed the inventive method, adds to the Arsenal of known fragments of leader peptides for microbiological synthesis of secreted target proteins in the yeast Saccharomyces cerevisiae and increases opportunities for the efficient synthesis of these proteins.

The inventive method allows for the secretion of target proteins in yeast S.cerevisiae variant of the Pro-region of the leader polypeptide protein PpPIR1 Pichia pastoris, the corresponding amino acid sequence of SEQ ID NO1, in combination with a variety of pre-regions.

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Zhang X, Kiechle F. (2001). Hoechst 33342-induced apoptosis is associated with decreased immunoreactive topoisomerase I and topoisomerase I-DNA complex formation. Ann Clin Lab Sci., 31(2):187-198.

Method of microbiological synthesis of the desired secreted protein by culturing in appropriate conditions, the yeast Saccharomyces cerevisiae, the secretion of the target protein in cells which directed the leader polypeptide, characterized in that the leader polypeptide includes a variant of the Pro-region of the leader polypeptide protein PpPIR1 Pichia pastoris, the corresponding amino acid in which sledovatelnot SEQ ID NO 1.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant human blood coagulability factor VIII with deletion of B-domain (hFVIII-BDD). Recombinant plasmid DNA pAP227 coding polypeptide with sequence hFVIII-BDD also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 2H5 producing recombinant hFVIII-BDD with highly stable yield at the level of about 20 IU/ml/24 h. Cultivation of cells-producers is performed in medium DME/F12 containing 2-4% of Fetal Bovine Serum, 1% of dimethylsulphoxide and 50 IU/l of insulin.

EFFECT: improvement of the method.

4 cl, 5 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

EFFECT: improvement of the method.

5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes compounds of labyrinthpeptins A1, A2, or A3 of formula (I) , where {A}, {B}, {C}, R1-R6, m and n have the values specified in the formula of the invention. Compounds are obtained at fermentation of Actinomadura namibiensis DSM 6313 strain under acceptable conditions in cultural environment till one or more compounds of formula (I) are formed. The invention proposes the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A2, and the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A1, as well as preprolabyrinthpeptins A1 and A2, and prolabyrinthpeptins A1 and A2. Labyrinthpeptins of formula (I) are used for therapy of infections caused by gram-positive bacteria, virus infections and/or neuropathic pain caused by inflammation.

EFFECT: improving therapy efficiency.

24 cl, 4 tbl, 20 ex

FIELD: biotechnology.

SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.

EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

16 cl, 2 dwg, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: plasmid genetic structure pOL-DsRed2 is produced, being built on the basis of a plasmid vector pIRES (Clontech), where fragments of cDNA of human genes OCT4 and LIN28 are placed, being connected with a nucleotide sequence coding P2A-peptide and gene cDNA, coding fluorescent protein DsRed2.

EFFECT: invention provides for simultaneous translation of human proteins OCT4 and LIN28 and fluorescent protein DsRed2 in production of induced pluripotent stem cells of a human being and animals in medicine and veterinary science.

3 cl, 1 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. The present invention discloses a codon-optimised gene coding the major capsid protein L1 of human papilloma virus which is able for effective expression of the major capsid protein L1 of human papilloma virus after transducing into a yeast cell. What is also described is an immunogenic macromolecule which is preferentially formed by the expression of the above codon-optimising gene coding the major capsid protein L1 of human papilloma virus in the yeast cell. What is also disclosed is using the above immunogenic macromolecule and composition containing the above immunogenic macromolecule.

EFFECT: presented group of inventions may be used in medicine for a human papilloma virus vaccine.

15 cl, 21 dwg, 12 tbl, 22 ex

FIELD: biotechnologies.

SUBSTANCE: microorganism-producent is cultivated in a suitable nutrient medium with subsequent extraction and treatment of target protein. At the same time the producent is yeast strain Saccharomyces cerevisiae, transformed with the expression vector, which contains an area of initiation of replication of endogenic 2-mcm plasmid of yeast Saccharomyces cerevisiae, and also a yeast promotor GAL1, which controls gene expression, including a DNA sequence SEQ ID NO: 1, coding a fused protein, the composite parts of which are aminoacid sequences of the protein E7-HSP70 and the protein of ubiquitin of yeast Saccharomyces cerevisiae, occupying within the fused protein an N-end position ending with a processing site, which separates it from the sequence of the protein E7-HSP70 and recognised natural ubiquitin-specific yeast proteinases. The yeast strain Saccharomyces cerevisiae VKPM Y-3853 - producent of the protein E7-HSP70 - is produced by transformation with the expression vector pPDX3U-E7-HSP70 of the yeast strain Saccharomyces cerevisiae D702.

EFFECT: group of inventions provides for high level of biosynthesis and yield of treated protein E7-HSP70, production of a water-soluble correctly processed protein E7-HSP70, principal absence of toxic and pyrogenic bacterial LPS in preparations of the protein E7-HSP70.

2 cl, 4 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: what is produced is the yeast strain Saccharomyces cerevisiae able to produce secreted human somatotropin. Said strain contains a promoter contolled DNA sequence coding mature human somatotropin fused with a leader peptide in the same reading frame. The leader peptide includes a double pro-site of α-factor of yeast Saccharomyces cerevisiae. It also can contain a triple pro-site of α-factor of yeast Saccharomyces cerevisiae, or a double pro-site of HSP150 protein of yeast Saccharomyces cerevisiae, or a combination of said pro-sites. A method for producing human somatotropin provides cultivation of the human somatotropin producer strain.

EFFECT: use of the invention provides higher end product yield.

2 cl, 1 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering and microbiological industry, as well as a genetic structure for exposing protein on the Yarrowia lipolytica yeast cell wall surface. The disclosed structure has a promoter, a terminator, a signal sequence, a selective marker, a nucleotide sequence which encodes an immobilised protein, and a nucleotide sequence which encodes an amino acid sequence containing an amino acid sequence selected from a group of sequences given in the list of sequences under number SEQ ID NO:1 or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6.

EFFECT: invention simplifies the process of purifying the protein produced by the cell and can be used in microbiological synthesis of proteins.

7 dwg, 11 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of producing recombinant spider-web proteins of orb-weaving spiders in yeast cells, fused proteins containing spider-web recombinant protein sequences of orb-weaving spiders, recombinant DNA encoding fused proteins, yeast host cells and expression vectors used to realise the method, as well as producer strains of recombinant proteins of orb-weaving spiders. The method involves constructing an expression vector containing a DNA sequence encoding recombinant spider-web proteins of orb-weaving proteins, fused with a sequence which encodes ubiquitin or an ubiquitin-lie protein SUMO of yeast Saccharomyces cerevisiae, which occupies the N-terminal position in the fused protein relative the recombinant spider-web protein, transformation of yeast cells with the obtained expression vector and expression of orb-weaving spider web protein in the transformed cells.

EFFECT: method increases production of recombinant spider-web protein during accumulation thereof in yeast cells in a water-soluble fraction in form of a protein which does not contain a hybrid component.

24 cl, 6 dwg, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of producing polyunsaturated fatty acids in seeds of transgenic plants. The method involves introducing into a plant, nucleic acids whose sequences code enzymes having Δ-6-desaturase, Δ-6-elongase, Δ-5-desaturase, Δ-5-elongase, Δ-4-desaturase and Δ-12-desaturase activity.

EFFECT: method increases content of polyunsaturated fatty acids in seeds of transgenic plants.

1 cl, 33 dwg, 24 tbl, 61 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology. The invention describes a yeast cell which contains an interference DNA construct. The construct comprises: a reporter gene under control of a first promoter; one or more in induced promoters called interference promoters selected and fixed in such a way that their activation induces transcriptional interference of the first promoter leading to a detectable decrease in expression of the reporter gene. The cell expresses: a first chimeric protein (Y-AD) formed by a transcription activation domain (AD) fused with a protein Y capable of interacting with at least a partner protein X; a second chimeric protein (X-DBD) formed by a first DNA-binding domain (DBD) fused with a second domain formed by protein X capable of interacting with protein Y, wherein the interaction of the two chimeric proteins X-DBD and Y-AD leads to formation of a functional transcription factor which activates the interference promoter(s). The invention discloses a method of intensifying a compound which inhibits interaction of the first protein X with the second protein Y.

EFFECT: invention enables detection of interruption of protein-protein interaction, as well as identification of alleles lacking on the level of interaction of proteins involved in the protein-protein interactions.

27 cl, 11 dwg, 4 tbl, 11 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is a Pachia pastoris PS 108 (pChIG) yeast strain, which is a producer of chicken interferon-gamma transformed by plasmid pChIG. Such plasmid pChIG, which facilitates biosynthesis of chicken interferon-gamma, has size of 8.19 thousand base pairs and consists of the following elements: - EcoRI-BamHI - a fragment of plasmid DNA of bifunctional bacterial-yeast vector pPIC9 with size of 7.75 thousand base pairs, - Bglll-EcoRI - a fragment with size of 0.44 thousand base pairs, which contains the coding part of the chicken interferon-gamma gene except the signal peptide coding region. The chicken interferon-gamma gene is obtained through a reverse polymerase chain reaction, whose matrix is RNA obtained from chicken peripheral blood mononuclear cells, induced by human recombinant interleukin-2 Roncoleikin ®.

EFFECT: invention enables obtaining chicken interferon-gamma with high efficiency.

3 cl, 2 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: there is offered method for identification and/or verification of inhibitors of receptor tyrosine kinases that involves application of a new test system which represents a yeast host cell containing an expression vector including a nucleic acid sequence that encodes fused protein essentially consisting of a complete cytoplasmic part of analysed receptor tyrosine kinase and the dimerisation domain and, if necessary, in addition including anchoring sequence for fused protein in a membrane wherein expression of fused protein conduces to termination of cell proliferation. The method provides production of specified host cells being in contact with a candidate compound and identification of inhibitors of tested tyrosine kinase activity as a result of cultivation on the assumption that inhibition of tyrosine kinase activity with a candidate compound causes restoration of proliferation process.

EFFECT: prospected application of the invention is related to development of selective therapeutic, including anticancer, agents.

13 cl, 5 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, more specifically to use of transformed cells Saccharomyces cerevisiae, which belong to a strain used in wine making, capable of high urea degradation in conditions of fermentation of grape juice, compared untransformed cells of the said strain. The cell is transformed by recombinant nucleic acid, which contains a sequence, which includes an open reading frame, homologic open reading frame DUR1,2 and a coding protein, with urea-degrading fermentation activity, and a promoter, suitable for mediating expression of a protein with urea-degrading fermentation activity, in conditions of fermentation of grape juice.

EFFECT: invention allows for obtaining wine, which cannot be distinguished from its organoleptic properties from wine obtained using untransformed cells.

13 cl, 7 dwg, 1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes two-stranded RNA modified with lipids and including antisense thread, the nucleotide sequence of which is complementary to target sequence in the target gene, and a sense thread, the nucleotide sequence of which is complementary to antisense thread. A two-stranded lipid is connected to the first nucleotide on the 5'-end of sense thread, either directly or through a linker. RNA has high stability to action of nuclease and is effectively absorbed with a cell, as well as generates excellent effect of RNA interference.

EFFECT: improvement of efficiency.

15 cl, 14 dwg, 3 ex

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