Application yeast strain komagataella pastoris as recipient for construction of producers of target protein

FIELD: biotechnology.

SUBSTANCE: application of yeast strain Komagataella pastoris RNCIM Y-727 as the recipient to construction of producers of target protein is characterised, optionally comprising introduction of mutations into it, providing the use of auxotrophic selective markers.

EFFECT: solution can be applied in preparation of recombinant proteins without the use of methanol as inductor of gene expression.

8 dwg, 4 tbl, 10 ex

 

Application of the strain of yeast Komagataella pastoris as a recipient for the design of the producers of the target protein

The invention relates to biotechnology and microbiological industry, in particular to receive the strain of the methylotrophic yeast Komagataella pastoris, suitable for construction on the basis of strains-producers, are able to synthesize the target protein.

Currently used in many pharmaceutical substances produced by microbial synthesis.

For the implementation of the expression, you must have the following system: the permitted use of a single-celled or multicellular organism, recipient, suitable for transformation vector comprising a DNA sequence encoding a target protein, and a vector for transformation, including all the necessary elements (selective markers, promoters, terminators, etc). Currently used expression systems can be divided into prokaryotic (bacterial) and eukaryotic, which are yeast cells, mineralnych fungi, insects and mammals.

Each system has its own technology and economic advantages and disadvantages. The use of bacterial expression systems, for example, the most commonly used in biotechnology expression system, using EQ what whether as a producer of a target protein, a recombinant strain of the E.coli bacteria, limits the range of proteins produced due to the inability to reproduce all post-translational modifications typical of eukaryotes, such as glycosylation, phosphorylation, formation of disulfide bonds between cysteine residues of CIS/TRANS isomerization of Proline, etc [1]. Also, when using the E.coli bacteria as a producer of the target protein, are often faced with the challenges of refolding (due to improper folding in the cytoplasm of cells and formation of insoluble Taurus inclusion), which makes impossible the use of E. coli for the biosynthesis of many proteins [1]. Synthesized by the cells of E.coli non-erythropoietin (glycoprotein, stimulator gamepause), the stability and activity of which depends directly on glycosylation, has a significantly lower stability compared to erythropoietin, secretively O-/N-glycosylated mammalian cells [2].

Use for secretion of target proteins in mammalian cells, providing all the necessary post-translational modification leads to an increase in production costs due to the need of using complex and expensive equipment and culture media for cultivation [3]. Using yeast as a producer of target recombinant proteins I have is a compromise, and in some cases the optimal solution. Yeast are representatives of eukaryotes and have the ability to all these post-translational modifications typical of eukaryotic cells, including secretion of proteins into the culture medium. Isolation and purification of the target recombinant product is greatly facilitated due to the fact that the number of endogenous secreted proteins in yeast is relatively small [3].

Interest in the production of recombinant proteins using expression systems based on the methylotrophic yeast, in particular, the genus Pichia, Komagataella or Hansenula [6] is based on several advantages of these systems as a scientific and technological point of view.

Culture methylotrophic yeast are grown in fermenters to a higher density, as compared with traditionally-used by the yeast Saccharomyces cerevisiae, which allows to obtain a higher level of production of the target protein [5]. The methylotrophic yeast have one of the most powerful in the nature of promoters. They are distinguished also an alternative mechanism for glycosylation of proteins to synthesize netcommunity target protein.

The most commonly used promoter for regulating expression of heterologous genes are AOJ (the promoter of the alcohol oxidase), GAP (promoter of glyceraldehyde 3-phosphate dehydrogenase), FLD (promoter formaldehyde dehydr genasi), FDH (promoter formate dehydrogenase), DHAS (dihydroxyacetone synthase) [9]. It is known that the promoters OH, FLD and FDH are induced in response to the presence in the medium of methanol or oleate. When culture growth on glycerol or glucose-containing medium, these repressed promoters [11]. When the depletion of the main carbon source, the promoter AOH is derepression state, while its activity is 1-2% of the activity in the induced state [12]. GAP is considered to be a constitutive promoter, i.e. induced during growth of the culture on many substrates, including glycerol, glucose.

A significant advantage of the methylotrophic yeast is the level and quality of N-glycosylation of proteins. Thus, in cells P.pastoris the size of the carbohydrate chains on average 8-12 residues of mannose, and the end balance is netcommunity α-1,2 mannosyl residue, whereas Saccharomyces cerevisiae usually secrete much more heavily glycosylated protein, containing up to about 100-150 residues per chain end balance which is highly immunogenic α-1,3 minasny the rest.

Using expression systems for various methylotrophic yeast, obtained a great variety of substances for the pharmaceutical industry: the surface antigen of hepatitis b (substance for vaccines), antistain, epidermal the ing growth factor, hirudin, α1-urokinase, endothelial growth factor, and others [3]. The first authorized the use of the product was registered by the FDA in December 2009.

As the closest analogue of the claimed invention consider the strain of methylotrophic yeast Komagataella pfaffii NRRL Y-48124. Strain GS115 (his phenotype-; Invitrogen, USA), which is its derivative, is widely used as an expression system in biotechnology [4]. Expression system based on this strain allows to obtain the target proteins with high yield, using induced by methanol promoter of the alcohol oxidase AH, the appeal of which lies not only in its activity, but in its strict regulation [12]. Unlike some strong promoters S.cerevisiae, which have a basal level of expression of repressive substrates, the promoter AOH completely repressed during growth on media containing such repressive substrates, such as glucose or glycerol, or ethanol [12]. Strict regulation of the promoter is an important factor in the expression of target genes encoding toxic to cells proteins.

When using strain GS115 derived products such as granulocyte-colony stimulating factor in the amount of up to 131 mg per 1 l of culture fluid [6] and recombinant hepcidin up to 100 mg per 1 l of culture fluid [7]Nedostatki this strain is the ability to Express the gene of the target protein, under the control of the promoter AH, only in the presence of the inducer methanol, which is toxic and easily inflammable substance[21], [22].

Sanitary-hygienic requirements to the organization and works with methanol impose serious restrictions on the use of this substance in biotechnological production [16]. In this regard, it is important to find a solution that, on the one hand, would allow use of the methylotrophic yeast for the production of the target protein; and on the other hand, would not use methanol as an inducer for the expression of genes of this protein.

The task of the invention is to expand the Arsenal of the methylotrophic yeast, suitable for construction on the basis of strains-producers, able to Express the target protein.

The problem is solved by applying a strain of yeast Komagataella pastoris VKPM Y-727 as a recipient for design on the basis of strains-producers, able to Express the target protein in the presence of the inducer methanol, and in his absence.

Obtaining and taxonomic characteristics of the strain .pastoris VKPM Y-727

Meeting the above requirements, the strain was found by screening in the all-Russian collection of industrial microorganisms (VKPM). The affiliation of strain .pastoris VKPM Y-727 to mate urovnem yeast of the genus Komagataella confirmed using growth tests on the respective substrates - methanol, glycerol, oleate, etc. in Addition to growth tests conducted molecular genetic comparison on the following markers: nucleotide sequence of domain D1/D2 26S subunit ribosomal RNA, the nucleotide sequence of the gene promoter of the alcohol oxidase (OH). Comparison with the corresponding nucleotide sequences of strain .pfaffii (NRRL Y-48124) and its derivative GS115 (Invitrogen, USA) showed homology at the level of 98%.

Morphological features:

Under cultivation at 28°C for 48 hours on agar YPD medium of the following composition (wt.%): peptone - 2, yeast extract - 1, glucose - 2, agar - 2, water - the rest, the cells are oval, 3-4 µm in diameter. Cells packouts. Budding true, multilateral. True mycelium form.

Colonies are the following:

1) on agar medium YPD colony light beige color with a smooth edge and a matte surface, a lenticular profile and a paste-like consistency;

Growth in YPD liquid medium composition (wt.%): peptone - 2, yeast extract - 1, glucose - 2, water - the rest at 28°C for 24 h cultivation - liquid cloudy, white precipitate, coagulation does not occur, parietal film does not form.

Physiological and biochemical characteristics: optional gone anaerobic. Temperature growth 20-33°C (optimum - 28°C). the pH of the environment to which steverivonia - a 4.8-7.4V (optimum - 6,0).

Assimilation of carbon sources: utilizes glucose, glycerol, methanol, oleate, sorbitol, rhamnose. Do not utilize galactose, xylose, arabinose.

Assimilation of nitrogen sources: utilizes amino acids, ammonium sulphate, ammonium nitrate.

Storage: at a temperature of -70°C in 30% aqueous solution of glycerol. Can be stored on agar medium YPD for 3 months at +4°C.

Regulatory elements

Sequenced following the sequence of the genome of strain .pastoris. VKPM Y-727:

The promoter of the alcohol oxidase OH strain .pastoris. VKPM Y-727 [SEQ ID No. 1] differs from the promoter of strain - the closest analogue is the following nucleotide substitutions: G->A at position 5, p->a in position 9, T->a in position 11, A->G at position 98, G->A at position 131, a deletion of G at position 132-133, a deletion in the position 170-171, S->T at position 201; G->A at position 206, a deletion of the MOP in position 311-312, T->a in position 354, A->G at position 380, G-A at position 482, C->T at position 568, T->a in position 582, A->T at position 611.

The promoter glyceraldehyde dehydrogenase GAP strain .pastoris. VKPM Y-727 [SEQ ID N2] is different from the promoter of strain - the closest analogue is the following nucleotide substitutions: G->A at position 61, S->T at position 86, A->C at position 180, S->T at position 201, T->C at position 202, And at position 209, A-G at position 211, T&g; With in position 240, a deletion of the ARTICLE in position 243-244, A->G at position 275; G->a in position 413, T->in position 450.

The invention is illustrated in the following figures.

Figure 1 Electrophoregram cell lysates of strain .pastoris VKPM Y-727 and K.pfaffii GS115

The arrows signal, which corresponds to the enzyme alcohol oxidase.

Track No. 1-7 correspond to

1. Molecular marker 70 kDa

2. The YPD medium, the strain .pastoris. VKPM Y-727

3. The YPD medium, the strain K.pfaffii GS115

4. Wednesday YPM, strain .pastoris VKPM Y-727

5. Wednesday YPM, strain K.pfaffii GS115

6. Wednesday YP, strain .pastoris. VKPM Y-727

7. Wednesday YP, strain K.pfaffii GS115

Figure 2 the Level of activity of beta-galactosidase produced by cells of strain .pastoris. VKPM Y-727 and strain K.pfaffii GS115

The chart shows measuring the activity of beta-galactosidase in the growth of cultures on three different environments: YP (imitating the depletion of the carbon source), YPM (containing methanol as inducer and carbon source), YPD (containing glucose as carbon source, which is a repressor of the promoter AOH).

On the vertical axis is the scale of activity in Milanovich units [Rose, Botstein et al.].

Dark columns correspond to the activity of the enzyme in the cell lysate of strain K.pastoris VKPM Y-727, light columns correspond to the activity of the enzyme in the cell lysate of strain K.pfaffii GS115.

On the chart bars are displayed with undertoe deviation on the measurement results of activity of the two clones.

Figure 3 Map of plasmid 81-29. The linearized fragment for transformation

HIS4-5' - 5'-region of the gene HIS4

HIS4-3' - 3'-region of the gene HIS4

URA3-pich - gene URA3

ORI - bacterial replicon

APRgene beta lactamase

MluI, BamHI sites of the restriction of the corresponding restricted

Tl6-fragm - items cloned from the field Tl6 retrotransposon TL1

Figure 4 Map of plasmid 85-011. The linearized fragment for transformation

ARG4-5' - 5'-region of the gene ARG4

ARG4-3' - 3'-region of the gene ARG4

URA3-pich - gene URA3

Prom - regulatory element of the gene URA3

ORI - bacterial replicon

APRgene beta lactamase

MluI, BamHI sites of the restriction of the corresponding restricted

Figure 5 Map of plasmid 86-011. The linearized fragment for transformation

ADE2-5' - 5'-region of the gene ADE2

ADE2-3' - 3'-region of the gene ADE2

URA3-pich - gene URA3

Prom - regulatory element of the gene URA3

ORI - bacterial replicon

APRgene beta lactamase

MluI, BamHI sites of the restriction of the corresponding restricted

Figa

Production of serum albumin (HSA), normalized by the volume of the culture fluid

On the vertical axis is the number of HSA in mg per 1 l of culture liquid.

Dark column corresponds to the number of HSA in the culture fluid of strain .pastoris VKPM Y-727. Light column corresponds to the number of HSA in the culture fluid of strain K.pfaffii GS115. displayed the standard deviation of the measurements from the two clones.

Figb

Production of serum albumin (HSA), normalized per unit of optical density

On the vertical axis is the number of HSA in mg per unit of optical density of the culture (OD=1 at λ=600 nm).

Dark column corresponds to the number of HSA in the culture fluid of strain .pastoris VKPM Y-727. Light column corresponds to the number of HSA in the culture fluid of strain K.pfaffii GS115. Straps shown the standard deviation of the measurements from the two clones.

Figa

Activity inulase produced the compared strains under the control of the GAP promoter

The vertical axis activity enolase in units of activity per 1 ml of the culture fluid per unit of optical density of the culture (OD=1 at λ=600 nm).

The dark column corresponds to the activity enolase in the culture fluid of strain K.pastoris VKPM Y-727. Light column corresponds to the activity enolase in the culture fluid of strain K.pfaffii GS115. Straps shown the standard deviation of the measurements from the two clones.

Figb

Activity inulase produced the compared strains under the control of the promoter AOH

The vertical axis activity enolase in units of activity per 1 ml of the culture fluid per unit of optical density of the culture (OD=1 at λ=600 nm).

The dark column corresponds to the activity enolase in the culture of the LM is bone strain .pastoris VKPM Y-727. Light column corresponds to the activity enolase in the culture fluid of strain K.pfaffii GS115. Straps shown the standard deviation of the measurements from the two clones.

Fig

The products of the surface antigen of hepatitis b (HBsAg)

On the vertical axis is the number of HBsAg in µg per 100 units of optical density of the culture (at λ=600 nm).

Dark column corresponds to the amount of HBsAg in the lysate of cells of strain .pastoris VKPM Y-727. Light column corresponds to the amount of HBsAg in the lysate of cells of strain K.pfaffii GS115. Straps shown the standard deviation of the measurements from the two clones.

Example 1. Confirmation of autoinduction promoter AOH in strain .pastoris VKPM Y-727

To confirm the induction of the promoter AOH in strain .pastoris VKPM Y-727 without the use of methanol, in terms of the depletion of the carbon substrate, the experiment is carried out; as control strains used strain .pfaffii GS115.

The analyzed strains were inoculated in tubes with medium three types of YPD with repressive promoter AOH carbon source is glucose, YPM the following composition (wt.%) (peptone - 2, yeast extract - 1, methanol and 0.5, water - else - inducing AOH carbon source is methanol, YP (wt.%) (peptone - 2, yeast extract - 1, water - else) without carbon source. In the logarithmic growth phase samples of cell lysate culture from CA the DOI tubes were analyzed by electrophoresis in denaturing reducing conditions in 12% polyacrylamide gel [Laemmli., 1970]. The result is shown in electrophoregram (figure 1).

From the data presented on electrophoregram, the signal intensity level of 70 kDa, which corresponds to the molecular weight of the alcohol oxidase [13], it is possible to judge the activity of the promoter of the alcohol oxidase in the appropriate culture conditions of the cells. Figure 1 shows that the gene AOH is not expressed during growth on medium containing glucose (track №2, 3), and is expressed during growth on medium with methanol in both strains (lanes 4 and 5), and is expressed in strain .pastoris VKPM Y-727 with the growth on the environment YP, i.e. in the absence of a carbon substrate. In the latter case, the expression level is 60-70% of the level of expression in the medium with methanol (induced), which confirms the activation of the promoter AOH in strain .pastoris VKPM Y-727 in growth conditions that mimic the depletion of the carbon substrate, without the use of methanol.

Example 2. Comparison of the strength of promoters OH from strain .pastoris VKPM Y-727 and a control strain K.pfaffii GS115

Each of the promoters OH of strains .pastoris VKPM Y-727 and .pfaffii GS115 independently of each cloned into a plasmid containing the gene Lac-Z (beta-galactosidase) to evaluate the strength of the promoter AOH each of the analyzed strains on the basis of the activity produced by engineered strains of the enzyme beta-galactoside is PS.

For this purpose constructed strains .pastoris Y-727/pINT-G418-AOX1Y727-LacZ and K.pfaffii GS115/pINT-G418-AOX1GS115-LacZ producers of beta-galactosidase.

Construction of plasmids

Constructed two plasmids: the plasmid pINT-G418-AOX1Y727-LacZ, designed for integration into the genome of strain .pastoris VKPM Y-727, and the plasmid pINT-G418-AOX1GS115-LacZ, designed for integration into the genome of strain K.pfaffii GS115. Both plasmids contain the elements necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. For carrying out the following procedure, transformation of yeast cells and the expression of a target gene Lac-Z (beta-galactosidase), the vector contains the following elements: gene neo - aminoglycoside 3'-phosphotransferase, the gene of resistance to the antibiotic G418 and kanamycin [18], under the control of the ADH1 promoter as a selective marker (ADH1 promoter was obtained by PCR, was used as a matrix genomic DNA of strain S.cerevisiae YBS618, primers prADH1-5'-aaagatctccatccttttgttgtttcc, prADH1-3'-cttgattgtatatgagata); gene Lac-Z [17]under the control promoter OH727(for strain Y727) or AOXGS115(strain GS115), cloned from strain .pastoris VKPM Y-727, either from strain K.pfaffii GS115, respectively, terminator OH. The promoter OH727and AOXS115 was obtained using PCR, was used as a matrix genomic DNA of strain .pastoris VKPM Y-727 or K.pfaffii GS115, respectively. Primers pr1_AOX1 (5'-ttcgtcgactaacatccaaagacgaaaggtt), pr2_AOX1 (5'-aaggtaccagatctagccatggtttggatccttcgaataattagttgttttttgatcttctcaa). Terminator OH was obtained using PCR, was used as a matrix genomic DNA of strain .pastoris VKPM Y-727 or K.pfaffii GS115, respectively, primers prAOXter-5' aatctcgaggattccagaatgccatttgcct, prAOXter3' gttgacgcgtgcacaaacgaacgtctcactt. Integrated part of the plasmid flanked by regions for embedding in a defined locus of the genome - endogenous promoter AOH. To obtain the linearized parts plasmids designed for the process of transformation, the original plasmid pINT-S-AOX1Y727-LacZ and pINT-G418-AOX1GS115-LacZ treated with restriction enzyme MluI (Thermo Fisher Scientific Inc.).

Transformation and cultivation

In the process of transformation according to standard Protocol [16] and selection of transformants got 2 producer strain with beta-galactosidase: .pastoris Y-727/pINT-G418-AOX1Y727-LacZ and control K.pfaffii GS115/pINT-G418-AOX1GS115-LacZ, in which the gene Lac-Z (beta-galactosidase) is under the control of promoters OH cloned from the corresponding compare strains. Then chose 2 clones for each strain. The inoculum of the analyzed clones were placed in test tubes with 5 ml of medium three types of YPD with repressive OH carbon source is glucose, PM - with inducing AOH carbon source is methanol, YP - without carbon source. The culture was grown in a rotary shaker-thermostat (250 rpm)at a temperature of 28°C. after 14 hours the cells were separated by centrifugation in a tabletop centrifuge at 3000 g and destroyed using glass beads to obtain a cell lysate [Miller et al.].

Assessment of the level of activity of beta-galactosidase

Assessment of the level of activity of beta-galactosidase contained in the cell lysate analyzed strains was carried out by a known method [Rose, Botstein et al.]. From table 1 and figure 2 data shows that the activity of beta-galactosidase in cell culture lysate of strain .pastoris Y-727/pINT-G418-AOX1Y727-LacZ grown on medium YP (without carbon substrate and inducer-methanol), is about 70% of the activity of beta-galactosidase from a lysate of the same culture grown on medium YPM (containing methanol). This allows us to conclude that the promoter AOH in cells .pastoris Y-727 activated in the presence of the inducer methanol, and in his absence, in contrast to the expression system control strain - the closest analogue K.pfaffii GS115. The level of synthesis of beta-galactosidase in both strains in the presence of methanol differs slightly in favor of strain K.pastoris VKPM Y-727.

Table 1
The activity of beta-galactosidase (ON)
Strain/EnvironmentYPYPMYPD
.pastoris Y-727/pINT-G418-AOX1Y727-LacZ1784,02668,00,0
1613,02124,00,0
Average1698,52396,00,0
Cf. square off.120,9284,70,0
K.pfaffii GS115/pINT-G418-AOX1GS115-LacZ621,02205,00,0
988,02541,00,0
Average804,52373,00,0
Cf. square off.259,5237,60,0

Note the p 3. Obtaining strain .pastoris Y727ura3mut

Construction of plasmids pURA3del

The vector contains the necessary elements for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. Gene URA3 obtained by PCR using as template genomic DNA of this strain, and primers pr1_URA3 (5'-gataacgcgtttgacgaattgactaaagttct), pr2_URA3 (5'-gataacgcgtttgacgaattgactaaagttct). Obtained by PCR gene cloned in a plasmid. Next, in the obtained plasmid was artificially disrupted reading frame of the gene URA3. After processing, the plasmid by restriction enzyme MluI received fragment, containing the flanks of the URA3 gene, so that the integration of this fragment into the genome of the transformed strain inaktivirovanie native URA3 gene, which resulted in the mutation of strain on the synthesis of uracil.

Transformation and selection of transformants

The original strain .pastoris VKPM Y-727 transformed received fragment of the plasmid pURA3del order to obtain mutant clones URA3 gene encoding orotidine-5-phosphate, a decarboxylase, an enzyme involved in the de novo synthesis of pyrimidine bases. The selection of mutant clones was carried out on selective cups YPD containing 5-ferrotitanium acid (5'-FOA), at a concentration of 500 μg/ml of agar medium. In the selection we selected 5 to the ons possessing the ability to grow in the presence of a given concentration of 5-ferrotitanium acid. Capacity growth in the presence of this substrate is connected with the probable mutation in the URA3 gene, the product of which is orotidine-5-phosphate decarboxylase, has no activity and no enzyme substrate (5'-FOA) to release toxic to cells fluorine. The resulting mutant had a phenotype ura-. The selected clones were tested for ability to grow on minimal agar medium YNB composition (wt.%) (0,17 yeast nitrogen base (Difco), 0.5 ammonium sulfate, 0.4 glucose, 2 agarose, the rest is water) with the addition of uracil at a concentration of 50 μg/ml of medium. All morphological features obtained mutants did not differ from clones of the original strain.

Example 4. Obtaining strain .pastoris Y-727ura3muthis4Δ

Construction of plasmids 81-29

Vector 81-29 contains the elements necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. HIS4 gene amplified using PCR. As the matrix used genomic DNA from strain .pastoris VKPM Y-727, primers: pr1_HIS4 (5'-ttcactcgtggatctataattgaacatgacatttcccttgctacct); pr2_HIS4 (5'-agatgccggttagatctatcgaat). Obtained by PCR gene cloned in the original plasmid. Gene URA3 obtained by PCR, COI is lsua as template genomic DNA of this strain, and primers pr1_URA3 (5'-gataacgcgtttgacgaattgactaaagttct), pr2_URA3 (5'-gataacgcgtttgacgaattgactaaagttct). Expression of cloned into a plasmid URA3 gene is under the control of the endogenous URA3 promoter. Gene URA3 with promoter flanked by sequences from the region that encodes a protein Tua retrotransposon TL1 cloned from yeast S.cerevisiae [19]. The resulting structure was inserted in the middle of the HIS4 gene (already cloned in the plasmid), so that she was surrounded by 5' and 3' regions of the gene HIS4. This design directs the integration into the HIS4 locus, after treatment of the plasmid with restriction endonucleases MluI, BamHI (Thermo Fisher Scientific Inc.) see (Figure 2). Thus, transformation of auxotrophic strain .pastoris Y-727ura3mutthe linearized fragment of the vector 81-29, as the result of homologous recombination between the endogenous locus HIS4 and given fragment, flanked by 5'- and 3'-regions of the HIS4 gene, is the integration of the linearized fragment of the plasmid with the replacement of native HIS4 gene.

The resulting linearized vector fragment 81-29 containing the URA3 gene, flanked by sequences from retrotransposon TL1, which in turn are flanked 5' and 3' regions of the HIS4 gene, cloned from strain .pastoris VKPM Y-727. The use of regions of retrotransposon TL1 provides vysielanie nucleotide sequence that is between them, and that is going to be used after the transformation.

Transformation of strain .pastoris Y-727ura3mut

Received auxotrophic strain .pastoris Y727ura3mut(example 3) transformed linearized fragment of the vector 81-29. In the homologous recombination during transformation is the deletion of the HIS4 gene. The genomes of the obtained transformants contain a fragment of the vector 81-29 with the gene URA3, instead replaced by the HIS4 gene. Selection of transformants was performed on the cups with minimal agar medium YNB with histidine at a concentration of 50 μg/ml of medium. A few selected transformants were inoculable in 5 ml of rich medium YPD. The culture grown for 24 hours, after which the culture was used as inoculum for new planting in the YPD medium. After three subcultures part of the cells were separated by centrifugation for cultivation on agar plates with YPD medium containing 5-torontobuy acid at a concentration of 500 µg/ml of medium for selection of those clones in which homologous recombination occurred between stroivshemsya in the HIS4 locus fragments Tl6, which led to the delegation of the gene URA3 concluded between the fragments Tl6 (Figure 3). As a result of recombination of the selected clone with his phenotype-ura-not able to grow on medium without histidine and uracil. Confirmed the ability of this clone to grow on minimal agar medium YNB containing uracil at a concentration of 50 μg/ml of CPE is s and the histidine at a concentration of 50 μg/ml

One of these clones, designated as strain .pastoris Y727ura3muthis4Δ. Received auxotrophic for histidine and uracil strain used for transformation vectors containing selective markers URA3 and HIS4, with subsequent selection of transformants on appropriate selective media.

Example 5. Obtaining strain .pastoris Y-727his4Δ

Received auxotrophic strain .pastoris s Y-727ura3muthis4Δ (example 4) was transformed with the PCR product containing the URA3 gene. For PCR the primers used were Pr1_URA3 (5'-gataacgcgtttgacgaattgactaaagttct) and Pr2_URA3 (5'-atttacgcgtactccttgagtctggtcaaa). As the matrix used for PCR of genomic DNA from strain .pastoris VKPM Y-727. Selection of transformants was performed on agar medium YNB with histidine at a concentration of 50 μg/ml of medium. As a result of transformation of the obtained clones with his phenotype-. Selected transformants were tested for ability to grow on minimal medium YNB with the addition of histidine at a concentration of 50 μg/ml of medium.

The resulting strain .pastoris Y-727his4Δ. Received auxotrophic for histidine strain was used for transformation vectors containing a selective marker HIS4, with subsequent selection of transformants on the appropriate selective medium.

Example 6. Obtaining strain .pastoris Y-727arg4Δhis4Δ

Construction of plasmids 85-011

Vector 85-011 contains the elements necessary for amplification of Vlatko E. coli: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. This vector contains the URA3 gene under the control of the native promoter and flanked by 5' and 3' regions of the gene ARG4, used to direct integration of this fragment directly in the ARG4 locus. 5' and 3' region ARG4 gene was obtained using PCR. As the matrix used in the genomic DNA of strain K.pastoris VKPM Y-727, primers:

pr1_ARG4 (5'-tctaacgcgtgtcgactaggtgttttaccctgattaga),

pr2_ARG4 (5'-ggatcctatctcgagtccggagatgctagcactaagatagctggtaataagtttaga),

pr3_ARG4 (5'-tagtgctagcatctccggactcgagataggatccatccattgactcccgttttga),

pr4_ARG4 (5'-gataacgcgtagatctcaattgtatgtactataacagttt).

Transformation and selection of transformants

Strain .pastoris Y727ura3muthis4Δ (example 4) transformed linearized fragment of the vector 85-011 (Figure 4). The fragment was obtained by processing vector 85-011 the restriction enzyme MluI (Figure 4). In the homologous recombination during transformation is the embedding vector fragment 85-011 in the ARG4 locus with replacement ARG4 gene (argininosuccinate), transformants have the phenotype ura+his-arg-. Selection of transformants was carried out on minimal agar medium YNB containing histidine at a concentration of 50 μg/ml and arginine at a concentration of 50 μg/ml of medium. Selected transformants possessed phenotype arg-his-.

Received auxotrophic for vistide is at and arginine strain .pastoris Y-727arg4Δhis4Δ suitable for producers by transforming vectors, containing a selective marker HIS4 or ARG4.

Example 7. Obtaining strain D. pastoris Y-727ade2Δhis4Δ

Construction of plasmids 86-011

Vector 86-011 contains the elements necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. This vector contains the URA3 gene under the control of the native promoter is flanked 5' and 3' regions of the gene ADE2 (enzyme phosphoribulokinase carboxylase), used to direct integration of this fragment directly into the ADE2 locus. 5' and 3' region of the gene ADE2 was obtained using PCR. As the matrix used in the genomic DNA of strain Komagataella pastoris Y727, primers: pr1_ADE2 (5'-tactaacgcgtgtcgacgaaagtaggcaaattagtttgt), pr2_ADE2 (5'-ctcgagtatggatcctccggagatcccgggacatgtgagctttgaaaatatctaatcgt), pr3_ADE2 (5'-acatgtcccgggatctccggaggatccatactcgagtcatcggtgttcctgtcaag), pr4_ADE2 (5'-agtaacgcgtagatcttgaaatagaaatatgattagaaaaaa).

Strain .pastoris Y-727ura3muthis4Δ (example 4) transformed linearized fragment of the vector 86-011 (Figure 5), which was obtained by processing vector 86-011 the restriction enzyme MluI (Figure 5). In the homologous recombination after transformation is the embedding vector fragment 86-011 in the ADE2 locus with replacement of the ADE2 gene (phosphoribosylglycinamide carboxylase), and the resulting transformants are ura phenotype+hs -ade-. Selection of transformants was carried out on minimal agar medium YNN containing histidine at a concentration of 50 μg/ml adenine at a concentration of 50 μg/ml of medium. Selected transformants possessed phenotype ade-his-. Received auxotrophic for histidine and adenine strain was used for transformation vectors containing a selective marker HIS4 or ADE2, with subsequent selection of transformants on the appropriate selective medium.

Received auxotrophic for histidine and adenine strain .pastoris Y-727arg4Δhis4Δ suitable for producers by transformation with vectors containing a selective marker HIS4 or ADE2.

Example 8. Obtaining strain .pastoris Y727his4Δ/pPH93-AOX1Y727-HSA - producer human serum albumin

Construction of plasmids

Expression of the integration vector rn-AOX1Y727-HSA contains the elements necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. For transformation of yeast cells and the expression of a target gene, the vector contains the following elements: HIS4 gene - histidinol dehydrogenase cloned from .pastoris VKPM Y-727, as a selective marker gene of the target protein is albumin (HSA) [SEQ ID N3]finding is different under the control of AOX1 promoter Y727[SEQ ID No. 1], cloned from .pastoris VKPM Y-727, terminator OH cloned from .pastoris VKPM Y-727. Integrated part of the plasmid flanked by regions for embedding in a defined locus of the genome - endogenous promoter AOH. To obtain the linearized part of the plasmid used for the transformation of yeast .pastoris VKPM Y-727, the original plasmid rn-AOX1Y727-HSA treated with restriction enzyme MluI.

Expression of integrative vector rn-AOX1GS115-HSA contains the same elements as the pPH93-AOX1Y727-HSA, except HIS4 gene - histidinol dehydrogenase cloned from .pfaffii GS115 (as a matrix used for PCR of genomic DNA of strain .pfaffii GS115, primers pr1_HIS4, pr2_HIS4), and promoter AOX1GS115cloned from .pfaffii GS115. To obtain the linearized part of the plasmid used for the transformation of yeast .pfaffii GS115, the original plasmid pPH93-AOX1GS115-HSA treated with restriction enzyme MluI.

Transformation

Getting producers secreted into the culture medium protein (human serum albumin, HSA) using strain .pastoris VKPM Y-727 and as a control strain .pfaffii GS115. As recipient strains used strain .pastoris Y-727his4Δ and strain .pfaffii GS115. The transformation of these strains linearized fragments of plasmids PP is 93-AOX1 Y727-HSA and pPH93-AOX1GS115-HSA, respectively, with subsequent selection of transformants on selective agar medium YNB without amino acids and ammonium sulfate, receive strains producing recombinant serum albumin.

As a result I received the following strains producers of serum albumin:

.pastoris Y727his4Δ/pPH93-AOX1Y727-HSA

.pfaffii GS115/pPH93-AOX1GS115-HSA

Cultivation

For the experiment chose 2 clones for each strain. The inoculum of the analyzed clones were placed in test tubes with 5 ml of medium YPgM, of the following composition (wt.%): peptone - 2, yeast extract - 1, glycerol, 0.5 to methanol and 0.5, water - the rest. The culture was grown for 62 hours in a rotary shaker-thermostat (250 rpm)at a temperature of 28°C. Every 24 hours spent induction with methanol, by aseptic addition of a 50% solution of methanol in a test tube, to a final concentration of 0.5%. After 62 hours, the biomass was separated by centrifugation in a tabletop centrifuge at 3000 g.

Measuring the concentration of albumin in the culture fluid was performed enzyme-linked immunosorbent assay on a solid substrate. As the substrate used standard tablet for enzyme immunoassay with pre-adsorbed human polyclonal antibodies to HSA. These antibodies, the conjugate - human polyclonals the e antibodies to HSA, labeled with horseradish peroxidase, standard for calibration, as well as the Chromogen TMB (3,3',5,5'-tetramethylbenzidine) was included in the standard set Albumin, Human, BioAssay ELISA Kit (manufactured by United States Biological, USA). The analysis was performed according to the Protocol described in the instructions to the set.

The yield of the target protein was 41,12 (+/-0,8) mg/l of culture fluid or of 1.93 (+/-0,2) mg / 1 optical unit) for strain .pastoris Y-727his4Δ and 41,19 (+/-1,4) mg/l of culture fluid or of 1.38 (+/-0,1) mg / 1 optical unit for a control strain K.pfaffii GS115.

The data presented in table 2 and figure 6 (a, b)indicate that, when normalized to the volume of the culture fluid strain producer .pastoris Y727his4Δ/pPH93-AOX1Y727-HSA is not inferior to the producer .pfaffii GS115/RRN-AOX1GS115-HSA, and when rationing on the optical unit even surpasses it in production.

Table 2
Productivity HSA
mg/lmg/1 PU
Y727his4Δ41,662,09
40,551,76
Average41,105 1,925
Article off.0,80,2
GS11540,231,31
42,14the 1.44
Average41,1851,375
Article off.1,40,1

Example 9. Receive producer inulase

Enolase - the enzyme used in the food industry to obtain fructose, by cleavage of the β-glycosidic linkages of inulin (polymaltose carbohydrates of plant origin).

Construction of plasmids

It was designed 4 expression of the integration vector, a different way of regulating gene expression of the target protein - enolase. Expression vector pINT-HIS4-GAPY727-INU contains the elements necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. For transformation of yeast cells and the expression of a target gene, the vector contains the following elements: HIS4 gene - histidinol dehydrogenase, as selective m is rcera, cloned from .pastoris PMBC Y727, the gene of the target protein - enolase cloned from Kluyveromyces marxianus [14], under the control of the GAP promoterY727cloned from .pastoris VKPM Y-727, terminator OH. The GAP promoter was obtained by PCR, was used as a matrix genomic DNA of strain K.pastoris VKPM Y-727, primers pr1_GAP (5'-aggaagcttttttgtagaaatgtcttggt), pr2_GAP (5'-ataggtacctgcagccatggtagatctttgatagttgttcaattgattgaaata).

To obtain the linearized part of the plasmid used for the transformation, the original plasmid pINT-HIS4-GAPY727-INU is treated with restriction enzyme MluI. Integrated part of the plasmid flanked by regions for embedding in a defined locus of the genome - endogenous promoter AOH. Expression vector pINT-HIS4-GAPGS115INU contains the same elements as the previous vector, except for selective marker, HIS4, which was cloned from .pfaffii GS115 (obtained using PCR, with matrix - genomic DNA of strain .pfaffii GS115, primers pr1_HIS4, pr2_HIS4), and promoter GAPGS115also cloned from .pfaffii GS115. The GAP promoter was obtained by PCR, was used as a matrix genomic DNA of strain .pfaffii GS115, primers pr1_GAP (5'-aggaagcttttttgtagaaatgtcttggt), pr2_GAP (5'-ataggtacctgcagccatggtagatctttgatagttgttcaattgattgaaata). When processed by a restriction enzyme MluI, the linearized fragment is also built into the locus of the genome OH.

Expression vector pINT-HIS4-AOX1Y727-INU contain the it elements, necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. For transformation of yeast cells and the expression of a target gene, the vector contains the following elements: HIS4 gene - histidinol dehydrogenase, as a selective marker cloned from .pastoris VKPM Y-727, the gene of the target protein - enolase cloned from Kluyveromyces marxianus [14], under the control of the AOX1 promoterY727cloned from .pastoris VKPM Y-727, terminator OH. To obtain the linearized part of the plasmid used for the transformation, the original plasmid pINT-HIS4-AOX1Y727-INU is treated with restriction enzyme MluI. Integrated part of the plasmid flanked by regions for embedding in a defined locus of the genome - endogenous promoter AOH. Expression vector pINT-HIS4-AOX1GS115INU contains the same elements as the previous vector, except for selective marker, HIS4, which was cloned from .pfaffii GS115 and promoter GAPGS115also cloned from .pfaffii GS115. When processed by a restriction enzyme MluI, the linearized fragment is also built into the locus OH.

Thus there was obtained 4 plasmids, two of which are:

pINT-HIS4-GAPY727-INU and pINT-HIS4-AOX1Y727-INU - designed for the transformatsii strain .pastoris Y-727his4Δ and differ only by the promoters (OH and GAP), the control expression of a target gene enolase in strain .pastoris Y-727his4Δ;

the other two pINT-HIS4-GAPGS115INU; pINT-HIS4-AOX1GS115INU - used for transformation of strain .pfaffii GS115 and differ only by the promoters (OH and GAP)that control the expression of target gene enolase in strain .pfaffii GS115.

Transformation

As recipient strains used strain .pastoris Y-727his4Δ and strain .pfaffii GS115, as a control. Thus the expression of a gene enolase regulated promoter AOH or GAP in each of the analyzed strains-producers.

The transformation of each of these strains linearized fragments of the plasmid pINT-HIS4-AOX1Y727-INU or pINT-HIS4-AOX1GS115INU respectively, followed by selection of transformants on selective agar medium YNB without amino acids and ammonium sulfate, receive the strains producing enolase: .pastoris VKPM Y-727/pINT-HIS4-AOX1Y727-INU and .pfaffii GS115/pINT-HIS4-AOX1GS115INU. Gene expression enolase is under the control of the inducible promoter AH.

The transformation of each of these strains linearized fragments of the plasmid pINT-HIS4-GAPY727-INU or pINT-HIS4-GAPGS115INU respectively, followed by selection of transformants on selective agar medium YNB without amino acids and ammonium sulfate, receive the strains producing enolase: .pstoris PMBC Y727/ pINT-HIS4-GAP Y727-INU and .pfaffii GS115/pINT-HIS4-GAPGS115INU. Gene expression enolase is under the control of the constitutive promoter GAP.

As a result, obtained the following strains-producers enolase:

Komagataella pastoris PMBC Y727/pINT-HIS4-GAPY727-INU

Komagataella pastoris PMBC Y727/pINT-HIS4-AOX1Y727-INU

Komagataella pfaffii GS115/pINT-HIS4-GAPGS115INU

Komagataella pfaffii GS115/pINT-HIS4-AOX1GS115INU

The determination of the activity of enolase

For the experiment chose 2 clones for each strain. The inoculate analyzed clone .pastoris Y727/pINT-HIS4-GAPY727-INU or .pfaffii GS115/pINT-HIS4-GAPGS115INU was placed in test tubes with 5 ml of YPD medium and inoculate clone .pastoris VKPM Y-727/pINT-HIS4-AOX1Y727-INU or .pfaffii GS115/pINT-HIS4-AOX1GS115INU - tube with 5 ml of medium YPgM.

The culture was grown for 40 hours in a rotary shaker-thermostat (250 rpm)at a temperature of 28°C. In the case of growth on the environment YPgM conducted induction with methanol after 24 hours of growth, by aseptic addition of a 50% solution of methanol in a test tube, to a final concentration of 0.5%. After 40 hours was separated biomass by centrifugation in a tabletop centrifuge at 3000 g.

Activity Sekretareva enolase was determined in the culture fluid, using as substrate sucrose. Inulase hydrolyzes β-glycosidic bond, resulting in a restoring carbohydrate - glucose. The number obrazovash is the action scene glucose allows to judge about the activity of the enzyme enolase. The amount of glucose measured by optical density (λ=492 nm) of the reaction mixture, after addition of 3,5-dinitrosalicylic acid, which restores up to 3-amino-5-nitrosalicylic acid, changing color [15]. Activity inulase presented in units. 1 unit of activity is that amount of enzyme that liberates 1 µmol of glucose per 1 min, normalized to 1 ml of the culture fluid and then to 1 optical unit (OD595culture.

From the obtained data are presented in table 3 and figure 7 a, b shows that the activity enolase in the case of expression under the control of the promoter AOH more than 2 times in the strain .pastoris VKPM Y-727/pINT-HIS4-AOX1Y727-INU, compared with the control strain .pfaffii GS115/pINT-HIS4-AOX1GS115INU. Conversely, in the case of the regulation of heterologous expression of the GAP promoter, the activity produced inulase more strain .pfaffii GS115/pINT-HIS4-GAPGS115INU, compared with K.pastoris Y727/pINT-HIS4-GAPY727-INU.

Table 3
Activity inulase
GAP promoterOH promoter
Y727his4Δ0,440,35
0,480,43
Average0,460,39
Article off.0,00,1
GS1151,110,18
0,970,16
Average1,040,17
Article off.0,10,0

Example 10. Getting a producer of intracellular protein surface antigen of hepatitis b virus

Construction of vectors pPH93-AOX1GS115-HBsAg; pPH93-AOX1Y727-HBsAg

Expression vector pPH93-AOX1GS115-HBsAg contains the elements necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. For transformation of yeast cells and the expression of a target gene, the vector contains the following elements: HIS4 gene - histidinol dehydrogenase cloned from strain GS115 with promotor part, as a selective marker; a gene of the target protein surface antigen VI the USA hepatitis b (HBsAg) [20], under the control of the promoter AH, terminator OH cloned from strain GS115. Integrated part of the plasmid flanked by regions for embedding in a defined locus of the genome - endogenous promoter AOH. To obtain the linearized part of the plasmid used for the transformation, the original plasmid rn-AOX1GS115-HBsAg is treated with restriction enzyme MluI.

Expression vector pPH93-AOX1Y727-HBsAg contains the elements necessary for amplification in E. coli cells: gene beta-lactamase (as a selective marker), bacterial replicon is to ensure a certain level of copy number plasmids. For transformation of yeast cells and the expression of a target gene, the vector contains the following elements: gene URA3 - orotidine decarboxylase cloned from the claimed strain with a promotor part, as a selective marker; a gene of the target protein - HBsAg, under the control of the promoter AH, terminator OH cloned from the claimed strain. Integrated part of the plasmid flanked by regions for embedding in a defined locus of the genome - endogenous promoter AOH. As a result of processing by the restriction enzyme MluI plasmids rn-AOX1GS115-HBsAg get the linearized part of the plasmid used for transformation of the recipient strain.

Transform the function

As recipient strains used strain .pastoris Y727ura3muthis4Δ and as a control strain .pfaffii GS115.

The transformation strain .pastoris Y727ura3muthis4Δ linearized fragment of plasmid pPH93-AOX1Y727-HBsAg, followed by selection of transformants on selective agar medium YNB containing histidine at a concentration of 50 μg/ml of medium, has been producing strains strain .pastoris Y727ura3muthis4Δ/pPH93-AOX1Y727-HBsAg synthesizing the surface antigen of hepatitis C.

The transformation strain .pfaffii GS115 linearized fragment of plasmid pPH93-AOX1GS115-HBsAg, followed by selection of transformants on selective agar medium YNB, has been producing strains strain .pfaffii GS115/pPH93-AOX1GS115-HBsAg synthesizing the surface antigen of hepatitis C.

The determination of the level of production of the surface antigen of hepatitis b virus

For the experiment chose 2 clones for each strain. The inoculum of the analyzed clones were placed in test tubes with 5 ml of medium YPgM. The culture was grown for 72 hours in a rotary shaker-thermostat (250 rpm)at a temperature of 28°C. Every 24 hours spent induction with methanol, by aseptic addition of a 50% solution of methanol in a test tube, to a final concentration of 0.5%. After 72 hours, the biomass was separated by centrifugation in the us is Olney the centrifuge, at 3000 g.

To determine the level of products used by enzyme immunoassay on a solid substrate. As the substrate used standard tablet for enzyme immunoassay with pre-adsorbed human polyclonal antibodies to HBsAg. This tablet, the conjugate is a mouse monoclonal antibodies to HBsAg, labeled with horseradish peroxidase and the Chromogen TMB (3,3',5,5'-tetramethylbenzidine) was included in the standard set of "Haastrup" (made in LLC "nearmedic plus") for determination of HBsAg in the serum.

Precipitated cells were destroyed in the buffer (10 mm phosphate buffer, 500 mm NaCl, 10 mm EDTA, 2 mm PMSF, 1% Tween 20, pH 7.2) using glass beads (Sigma) on the cage FastPrep 24 (Mr). Analyzed the amount of HBsAg in the resulting lysate. As a standard for calibration used industry standard sample HBsAg hepatitis b virus (cat. No.-331, LLC "Scientific-production Association Diagnostic system"). The amount of HBsAg in the total lysate of the clones was determined by the intensity of the absorption of the reaction mixture at a wavelength of λ=492 nm. Quantitative data normalized to 100 optical units and is shown in Fig and in table 4, from which it is clear that the products of the surface antigen of hepatitis b virus is similar in both strains compared within measurement error.

Table 4
Productivity HSA
µg/100 PU
Y727his4Δ87,2
to 91.1
Average89,15
Article off.2,8
GS11593,5
84,4
Average88,95
Article off.6,4

Thus, confirmed the effective application of the strain of the methylotrophic yeast .pastoris VKPM Y-727 as strain-recipient to construct producers that are able to synthesize the target protein. The strain has the following characteristics corresponding or superior characteristics of the strain - the closest analogue is:

- suitable for synthesis of various target proteins, in particular inulase, serum albumin, surface antigen hepatitis b virus, beta-galactosidase, which is confirmed by examples 2, 8, 9, 10; this can be used as induced (OH)and constitutive (GAP) p is amatory (examples 2, 8, 9, 10);

unlike the closest analogue, allows regulated promoter OH the synthesis of the target protein in the presence of the inducer methanol, and in its absence, with output up to 70% of the level of synthesis of this protein in the presence of methanol (examples 1, 2);

- makes it easy to get traditionally used in genetic engineering of auxotrophic mutants, which is confirmed by examples 3, 4, 5, 6, 7.

The list of scientific and technical information

1. Daly R, Milton TW Hearn (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit. 18: 119-138.

2. Narhi LO, Arakawa T, Strickland TW. (1991). The effect of carbohydrate on the structure and stability of erythropoietin. J. Biol. Chem. 266: 23022-23026.

3. Macauley-Patrick S, Fazenda ML (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22: 249-270.

4. Balamurugan V, Reddy GR (2006) Pichia pastoris: A notable heterologous expression system for the production of foreign proteins - Vaccines. Indian Journal of Biotechnology (6): 175-186.

5. Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl. Environ. Biotechnol. 54(6): 741-750.

6. Kurtzman CP (2009) Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. J Ind Environ Biotechnol 36: 1435-1438.

7. Bhatacharya P, Pandey G (2007) Production and purification of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) from high cell density cultures of Pichia pastoris. Bioprocess and Biosystems Engineering 30(5): 305-312.

8. Zhang H, Yuan QP (2007) Cloning and secretion expression of hepcidin in Pichia pastoris. Sheng Wu Gong Cheng Xue Bao 23(3): 381-385.

9. Yurimoto H (2011) Yeast Methylotrophy: Metabolism, Gene Regulation and Peroxisome Homeostasis. Hndawi Publishing Corporation International Journal of Microbiology Article ID 101298.

10. Conde R, Cueva R (2004) A Search for Hyperglycosylation Signals in Yeast Glycoproteins. The journal of biological chemistry (42): 43789-43798.

11. Hartner A, Glieder A (2006) Regulation of methanol utilization pathway genes in yeasts. Microbial Cell Factories 2006, 5: 39.

12. Tschopp JF, Brust PF, Cregg JM (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 15: 3859-3876.

13. Cregg JM, Barringer KJ (1985) Pichia pastoris as a Host System for Transformations. MOLECULAR AND CELLULAR BIOLOGY, Dec. 1985, p.3376-3385.

14. Brevnova EE, Kozlov became popular Strain of Saccharomyces cerevisiae producing fructose from inulin, 1998, Biotechnology, 1, 12-20.

15. Berezin IV, Rabinovich ML Study of Applicability of quantitative kinetic spectrophotometric method for glucose determination, 1997, Biokhimiya, 42: 1631-1637.

16. Shixuan Wu, Geoffrey J. Letchworth High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol, BioTechniques, 2004, 36: 152-154.

17. Rose M, Botstein D Construction and use of gene fusions lacZ (b-galactosidase) which are expressed in yeast. Meth. Enzymol. 101: 167-180.

18. Oka A Sugisaki H, Takanami M Nucleotide sequence of the kanamycin resistance transposon Tn903 JOURNAL J. Mol. Biol. 147 (2), 217-226 (1981).

19. Marchenko A.N., Kozlov became popular, Swarsensky E.V., Benevolensky SV using the N-Terminus of the protein Tua to create a hybrid virus-like particles. Biotechnology 2001, No. 2, 3-11.

20. Capregen SE, Arman I.P., N. Granovsky. "Optimization of gene expression of the surface antigen of hepatitis b virus in yeast". Mol. genetics, Microbiology, Virology 1990, 5, p.17-20.

21. The resolution of the Chief state sanitary physician of Russian Federation from July 12, 2011 N 99 Moscow "On approval of the JV 2.3.3.2892-1 "the Sanitary-hygienic requirements to the organization and works with methanol".

22. State sanitary and epidemiological regulation of the Russian Federation 4.1. Control methods. Chemical factors, the determination of concentrations of chemicals in the air. Gas chromatographic determination of methanol in the air. HOWTO MUK a-01 Issue 2, Ministry of health of Russia, Moscow 2002.

Application of the strain of yeast Komagataella pastoris VKPM Y-727 as a recipient for the design of the producers of the target protein, optionally including the introduction of mutations, providing the use of auxotrophic selective markers.



 

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2 dwg, 4 ex

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: medicine.

SUBSTANCE: invention relates to biochemistry. A method of immunoassay of human protein CXCL1 is described. Human CXCL1 or its fragment is measured in a sample with application of two or more types of monoclonal antibodies to human CXCL1 or their fragments. Each of two or more types of the monoclonal antibodies to human CXCL1 or their fragments specifically identifies any of regions of a sequence of amino acid sequences, represented in SEQ ID NO:1-3, which represent partial sequences of an amino acid sequence, constituting human protein CXCL1. Two or more types of the monoclonal antibodies to human CXCL1 or their fragments specifically identify regions of the sequence, different from each other. Claimed are the monoclonal antibodies or their fragments, each of which specifically identifies any region of the amino acid sequence, represented in SEQ ID NO:1-3, and has a new amino acid sequence.

EFFECT: invention makes it possible to determine human protein CXCL1 with high sensitivity.

15 cl, 9 dwg, 1 tbl, 21 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.

EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.

3 cl, 6 ex, 1 tbl, 14 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry and represents method of obtaining L-amino acid - L-lysine, L-threonine, L-asparagine, L-aspartic acid, L-methionine or L-isoleucine, which includes cultivation of bacteria Escherichia coli in medium for obtaining and accumulation of L-amino acid and collection of L-amino acid. Bacterium is modified in such a way as to increase expression of its gene gltP or gene gltS.

EFFECT: invention makes it possible to increase output of target L-amino acid.

12 cl, 5 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: invention refers to the field of biotechnology and food industry. Presented is a barley plant that yields grain and is homozygotic in at least two loci for the genetic variations having been bred, representing: a) allele wherein most of or all the genes coding B-hordein in Locus Hor2 are removed, and b) mutant allele in the barley Locus Lys3 so that the grain contains neither B-, nor C- hordeins, the said genetic variations present in Lines Riso 56 and Riso 1508 barley accordingly; absence of B-hordeins is to be revealed by absence of amplified DNA using primers: 5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3', 3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3', while absence of C-hordeins is to be revealed by absence of the 70 kDa strip during study of the grain alcohol-soluble extract by means of SDS-PAGE. Additionally presented are: barley grain cropped from the said plant; B- and C-hordein-free products produced from the said grain such as flour, malt and beer. Additionally described are methods for production of food products barley (flour, whole-grain flour, starch, malt) and beverages using grain cropped from the barley plant having the above characteristics. Proposed is a method for identification of barley grain suitable for production of a malt-based food product and/or beverage suitable for consumption by a person suffering from gluten-sensitive enteropathy which method includes: a) production of one or more materials: i) sample of a plant capable to yield the said grain, ii) grain, iii) malt produced from the grain, and/or iv) extract of the said grain; b) analysis of Stage a) material for presence of at least one hordein and/or at least one hordein-coding gene with selection of grain having the gene pattern of the above plant.

EFFECT: invention allows to manufacture B- and C-hordein-free malt-based food products or beverages.

27 cl, 14 dwg, 10 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed is separated chimeric polynucleotide for amplification of production of heterologous protein of interest, which contains polynucleotide sequence of promoter SigA or SigH, functionally connected with polynucleotide, coding protein YmaH, with chimeric polynucleotide connecting sequence, which by, at least, 90% is identical to SEQ ID NO: 1, 2, 3 or 13. Also described are: expression vector, containing claimed nucleotide structure, and host cell Bacillus for production of heterologous protein of interest, which contains said vector. Claimed is method of obtaining modified Bacillus cell, including transformation of host cell of Bacillus-producent of heterologous protein of interest with said vector; and growing said modified cell in optimal conditions. Described is method of obtaining protein of interest in modified Bacillus cell, where method includes cultivation of said host cell; and growing said modified Bacillus cell in optimal conditions. Also described is method of amplification of expression of heterologous protein from Bacillus of interest includes obtaining said modified Bacillus cell; growing modified Bacillus cell in optimal conditions; and expression of said protein of interest in modified Bacillus cell, where expression of said heterologous protein of interest in modified Bacillus cell is amplified in comparison with expression of said protein of interest in said parent Bacillus host-cell.

EFFECT: invention makes it possible to increase output of target protein due to superexpression of protein YmaH.

30 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to methods of targeted change of duplex acceptor DNA-sequence and improvement of efficiency of targeted mutagenesis in plant protoplasts. Claimed methods include introduction into protoplasts of plant cells of single-stranded mutagenic oligonucleotide, which in its composition has, at least, one wrong nucleotide with respect to duplex acceptor DNA-sequence, which is to be changed with application of PEG-mediated transformation.

EFFECT: invention makes it possible to increase frequency of targeted mutagenesis.

10 cl, 3 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and discloses novel diacylglycerol acyltransferase. The invention also relates to a polynucleotide which codes diacylglycerol acyltransferase, an expression vector and a transformant such as yeast or fungus, as well as a method of preparing a composition of lipids or fatty acids using the transformant.

EFFECT: invention enables to obtain a novel enzyme which is suitable for efficient production of lipids and fatty acids.

13 cl, 5 dwg, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns preparing a genetic construct providing a synthesis of p35d recombinant protein in Escherichia coli cells. There are presented: recombinant plasmid DNA pQE-p35d providing the synthesis of p35d recombinant protein of cowpox virus and containing in accordance with physical and genetic map presented on Fig. 2: pQE30 plasmid vector, a fragment coding MRGSHHHHHHG oligopeptice and a fragment of 17 base pairs, coding a fragment of p35 protein of cowpox virus within 1 to 239 amino acid residues (Fig.1a); Escherichia coli XL1Blue/pQE-p35d B-1252 bacterial strain that is a producer of p35d recombinant protein of cowpox virus, containing recombinant plasmid DNA pQE-p35d deposited in the Collection of Bacteria, Bacteriophages and Fungi of FBUN GNTs VB Vector, registration No. B-1252, and p35d recombinant protein of cowpox virus.

EFFECT: solutions may be used to engineer the test systems and to prepare orthopoxvirus split vaccines.

3 cl, 7 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of obtaining a recombinant antigen G2 of hantaviruses. The disclosed method is characterised by that the DNA structure pGHF, which encodes a fused protein of three parts, where N-terminal position is occupied by a green fluorescent protein GFP, central position is occupied by a peptide of 73 amino acid residues with the amino acid sequence SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and the C-terminal position is occupied by a mini-domain Foldon of coliphage fibritin JS98C3 (dwg 2), is introduced into E. coli cells; cells transformed by this structure are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifuging, the product of expression in the form of inclusion bodies is solubilised with methylated spirit, chromatography is carried out and the obtained product is used to detect specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: disclosed solution improves repeatability and sensitivity of immunoenzymatic assay when diagnosing hemorrhagic fever with renal syndrome.

7 dwg

FIELD: biotechnologies.

SUBSTANCE: invention refers to biotechnology and gene engineering and represents recombinant pHisTevTSIB0821 plasmid for expression in Escherichia coli cells of TSIB_0821 prolidase from Thermococcus sibiricus archean. The proposed plasmid includes NdeI/SalI-fragment of pET-22b(+) (Novagen) plasmid and a DNA fragment with the size of 1196 pairs of bases, which contains a fused gene consisting of the following structural elements: nucleotide sequence coding 6-histidine tag, nucleotide sequence coding the site of recognition/decomposition of TEV protease, and nucleotide sequence of TSIB_0821 gene, which are connected so that at their biosynthesis in E. coli cells a continuous reading frame can be maintained. E. coli Rosetta(DE3) strain is obtained, which is transformed with the above plasmid, - a producer of chimeric protein including amino acid sequence of TSIB_0821 prolidase, fused on N-end with the 6-histidine tag and the site of recognition/decomposition of TEV protease. A growth and induction method of a producer strain and a method for separation and cleaning from the obtained biomass of functionally active recombinant TSIB_0821 prolidase including the following technological process: two metal affine chromatographies, gel filtration, TEV protease treatment, dialysis, and concentration, have been developed.

EFFECT: invention allows obtaining recombinant prolidase that is similar as much as possible as to structure to its natural equivalent with high and stable yield, level of cleaning and functional activity.

3 cl, 3 ex

FIELD: biotechnology.

SUBSTANCE: hybrid proteins GFN80 and GFN100 are formed based on recombinant human interferon alpha-2 fused on the N-terminus with the amino acid sequence of polypeptide S(G4S)16 or S(G4S)20, respectively. The strains of producer Saccharomyces cerevisiae RNCIM Y-3927 and Saccharomyces cerevisiae RNCIM Y-3928 are produced by recombinant method. The strains are used in the method of production of the hybrid protein GFN80 and GFN100, which comprises culturing under suitable conditions of yeast cells transformed by the expression vector, which contains the region of replication initiation of endogenous 2-micron plasmid of yeast Saccharomyces cerevisiae, and the promoter of yeast GAL1 controlling the expression of the gene comprising the DNA sequence SEQ ID NO:1 or SEQ ID NO:2, respectively, followed by isolation of the hybrid protein from the culture fluid.

EFFECT: invention enables to produce the hybrid recombinant human interferon alpha-2 with the prolonged action in the body of animals.

5 cl, 7 tbl, 15 ex

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