RU2150501C1 - Strain of yeast saccharomyces cerevisiae vkm cr-349d as producer of human epidermal growth factor - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: invention relates to producing recombinant human epidermal growth factor. Recombinant strain of yeast, Saccharomyces cerevisiae, as producer of human epidermal growth factor is prepared by transformation with recombinant plasmid YEp4OAB directing synthesis of human epidermal growth factor and its export in culturing medium. The strain Saccharomyces cerevisiae is deposited in VKM at number VKM CR-349D. Strain is able to produce human epidermal growth factor in the amount 10 mg/l of producer culture at density 5 x 10⁸ cells/ml. Recombinant human epidermal growth factor corresponds to the natural human epidermal growth factor by its biological and physical-chemical properties. EFFECT: production of biologically active human epidermal growth factor, high yield of factor. 1 tbl, 4 dwg, 5 ex

Description

 The invention relates to biotechnology, in particular to genetic engineering, and is a recombinant plasmid DNA that determines the synthesis and secretion of human epidermal growth factor (hEFR) in yeast cells and the yeast strain Saccharomyces cerevisiae - producer of hEFR.

 The invention can find application in the microbiological industry and medicine, in particular for the creation of new anti-burn and wound healing agents, for the diagnosis of malignant diseases associated with hyperproduction of the chEFR receptor, as well as in the creation of new methods for targeted delivery of certain therapeutic agents to target cells expressing specifically chEFR receptor.

 CHEFR is a peptide hormone produced by various tissues and found in almost all biological fluids of the human body. CHEFR is synthesized in the form of a precursor with a length of 1217 amino acids and undergoes further pro-proteolytic processing to form a mature protein with a characteristic size of 53 and with three internal disulfide bonds [1]. CHEFR acts on tissues containing the corresponding receptor on its surface and stimulates their growth and division. The most potent mitogen in vivo and in vitro hEFR is for epithelial and connective tissue cells. A detailed study of the spectrum of EGF effects indicates the possible ways of its biotechnological application as a wound healing, cytoprotective, antiulcer drug, etc. [2-7].

To date, recombinant plasmid DNAs are known that provide for the expression of hEFR in the cells of the bacteria Escherichia coli, B. subtilis and the yeast S. cerevisiae and Pichia pastoris [8–11]. The use of the secretory pathway of microorganisms to obtain biologically active peptides of mammals in comparison with other methods of their biosynthesis (for example, in the form of hybrid proteins) has several advantages, namely:
- in the process of secretion, the correct maturation and folding of the recombinant protein occurs;
- secreted protein accumulates in a significant amount in the extracellular space of the recombinant strain and is protected from the action of intracellular proteases, which greatly facilitates its subsequent isolation and purification.

 The advantage of saccharomycetes yeast as producer strains of secreted biologically active mammalian peptides is that these microorganisms are safe for humans, free of endotoxins and allergens present, for example, in the cell walls of Escherichia coli bacteria. The obtained preparations of recombinant CHEFR can be used for scientific, therapeutic and cosmetological purposes, as well as as components of culture media for animal cell cultures.

 The aim of the invention is to obtain a recombinant strain of yeast that produces mature and biologically active hEFR in high yield in secreted form. This goal is achieved by the fact that a recombinant plasmid YEp40AB was constructed, which ensures the synthesis and secretion of hEFR in yeast cells.

 To achieve this goal, it is also proposed a strain of Saccharomyces cerevisiae 20B12 VKM CR-349D containing recombinant plasmid DNA YEp40AB.

 The essence of the recombinant plasmid YEp40AB, whose genetic map is shown in FIG. 1 consists in the fact that it contains a synthetic CHEFR gene integrated into the expression of the yeast vector YEpGPD / SEC, which in turn contains the replicon of the plasmid pUC18, replicon 2m of the yeast DNA, the yeast gene GPD1 promoter, the signal sequence for the secretion and processing of yeast alpha pheromone alpha factor, yeast PGK gene terminator, yeast TRP1 gene.

Plasmid YEp40AB consists of the following elements:
- fragment EcoRI "stupid" -HindIII size 2.6 TPN plasmids pUC18
- fragment EcoRI "dull" -PstI size of 2.0 TPN 2 m DNA of yeast
- a fragment of EcoRI "dull" -PstI size 0.9 TPN carrying the TRPI gene of yeast
- a 1.25 kb HindIII EcoRI fragment of HdIII containing the promoter of the yeast GPD1 gene fused to the preprosegment of the yeast MFa1 gene;
a 0.3 kb ClaI-HindIII fragment containing yeast PGK1 gene terminator
- 0.15 kb EcoRI-ClaI fragment containing the CHEFR gene.

 The molecular weight of the plasmid YEp40AB is 4.5 MD, which corresponds to a size of 7.2 TPN.

 The number of copies of the obtained plasmid is 10-15 per yeast cell.

 YEp40AB plasmid DNA contains unique sites for restriction enzymes AatII, BglII, ClaI, EcoRI, two sites for restriction enzymes PvuII, XbaI, the positions of which are indicated in table. 1. This fact was established using restriction analysis.

 Plasmid YEp40AB is replicated in E. coli cells due to pUC18 replicon. In yeast cells, plasmid replication is provided by a replicon of 2 mcm yeast DNA. The synthesis of CHEFR in yeast cells is carried out under the control of the constitutive yeast GPD1 promoter and the processing and secretion signals of the MFα1 gene and is achieved by culturing the transformed strain in ordinary selective and semi-selective media.

Plasmid YEp40AB contains the genes:
- a modified synthetic hEFF gene that encodes the synthesis of this protein in a yeast cell. This gene, introduced between the EcoRI and ClaI cleavage sites, has the nucleotide sequence shown in FIG. 2. This sequence encodes a mature hEFR. In front of this gene is the synthetic LysArg sequence, which is a specific processing site for the yeast endoprotease KEX2, which is part of the preprosegment of the yeast MFa1 gene.

- bla-gene, providing synthesis of b-lactamase and affecting the resistance of strains of E. coli carrying the plasmid to ampicillin
- TRPI gene providing tryptophan prototrophy of transformants of the recipient strain of the yeast carrying the plasmid.

 The resulting product is a mature CHEFR containing amino acid residues.

 The mitotic stability of the plasmid YEpGPD1 / EGF in the strain S. cerevisiae 20B12 with growth under selective conditions is 90%.

 The hEFF producing strain is obtained by transforming the Saccharomyces cerevisiae 20B12 [a / a trp1 pep4-3] yeast cells of the proposed recombinant plasmid DNA. The choice of the strain is due to the fact that it carries the only auxotrophic mutation in the trp1-289 gene, which allows selection of transformants by the plasmid YEp40AB according to the trp1 marker, and also because it is defective in the synthesis of vacuolar proteases, which significantly affects the yield of synthesized heterologous proteins.

The transformed clone 20B12 / YEp40AB obtained after selection is capable of producing hEFR upon cultivation. The level of hEFR synthesis in the constructed strain is 10 mg / L with a culture titer of 5 • 10 ⁸ cells / ml, which follows from the data on the determination of hEFR in the supernatants of the culture medium of the producer strain using enzyme immunoassay.

The resulting yeast strain carrying the YEp40AB plasmid is characterized by the following common features:
Morphological signs. Cells are round to oval in shape, budding when dividing.

 Cultural signs.

 Cells grow well on commonly used culture media. The generation time is about 90 min in a liquid YNB medium. On 2-2.5% nutrient agar "Difco" round, smooth, white colonies with even edges are formed. When grown on liquid YNB and YEPD media, intense even turbidity forms.

 Physiological and biochemical characteristics.

The optimum cultivation temperature is from 28 to 30 ^o C, the optimum pH is 6.6. The source of carbon is mainly glucose, in some cases, ethanol. The source of nitrogen is organic compounds (in the form of peptone, amino acids).

The method for producing the recombinant plasmid YEp40AB is illustrated by the following examples:
Example 1. Construction of an intermediate plasmid pUC9 / hEGFdBam
Plasmid pUC9 / hEGF is a pUC9 vector containing a synthetic human epidermal growth factor gene [12], cloned between the EcoRI and BamHI sites of the pUC9 polylinker.

2 μg of pUC9 / hEGF plasmid DNA was linearized using BarnHI restriction enzyme by incubation for 120 min at 37 ^° C in high salt buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM DTE, 10 mM MgCl2) with 10 enzyme units . Then, 2 μl of a solution containing all four deoxynucleotide triphosphates at a concentration of 2 mM each and 2 units of the Klensky fragment of DNA polymerase I was added to the reaction mixture and incubation continued for another 30 min at room temperature.

 The PUC9 / hEGF plasmid DNA linearized and blunted at the BamHI site was freed from the proteins present and purified using the GeneClean II kit (Bio101 Inc.) according to the manufacturer's instructions.

100 ng of the obtained preparation in a volume of 20 μl were ligated in a mixture of 50 mM Tris-HCl pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP with the addition of T4 ligase (2.5 units) for 3 hours at 16 degrees WITH.

 200 μl of competent cells of E. coli strain JM110 prepared in a standard way [13] were transformed with 10 μl of ligase mixture and transformants were selected on nutrient agar plates containing 50 μg / ml ampicillin.

1 мкг плазмидной ДНК инкубировали 2 часа при 37^oC в 1х низкосолевом буфере с 2 единицами рестриктазы ClaI и полученные препараты анализировали в 1% агарозном геле. Те плазмиды, которые образовывали 3 тпн фрагмент ДНК, соответствовали правильной конструкции. Эта плазмида была обозначена как pUC/hEGFdBam.Plasmid DNA from the obtained clones was isolated using the alkaline method [13]. All plasmid DNAs were checked by restriction analysis for the appearance of an additional site for the ClaI restriction enzyme formed by "filling" the BamHI site, as follows:

1 μg of plasmid DNA was incubated for 2 hours at 37 ^o C in 1x low-salt buffer with 2 units of restriction enzyme ClaI and the resulting preparations were analyzed on a 1% agarose gel. Those plasmids that formed a 3 bp DNA fragment corresponded to the correct design. This plasmid was designated as pUC / hEGFdBam.

Example 2 Construction of Recombinant Plasmid YEp40AB for Expression and Secretion of hEFR in Yeast Cells
The construction was carried out in accordance with the circuit shown in FIG. 1.

 Plasmid YEpGPD / SEC [14] is a vector for the expression and secretion of foreign gene products in yeast.

To obtain a “vector”, 10 μg of YEpGPD / SEC plasmid DNA was incubated for 2 hours at 37 ^° C in high-salt buffer with 10 units of EcoRI in a volume of 20 μl, the completeness of hydrolysis was monitored by electrophoresis in 1% agarose gel. The mixture was then heated for 15 minutes at 70 ^° C to inactivate the restriction enzyme and was diluted 5 times with 1x low-salt buffer. Next, 10 ClaI restriction enzyme units were added and incubation continued for another 2 hours at 37 degrees.

 The mixture was deproteinized using a Gene CleanII kit and DNA was dissolved in 20 μl of TE buffer.

 To obtain a “fragment”, 30 μg of the plasmid PUC9 / hEGFdBam was sequentially digested with restriction enzymes EcoRI and ClaI as above, proportionally increasing the initial volume of the incubation mixture three times. After deproteinization using the Gene CleanII kit, DNA was dissolved in 40 μl of TE buffer and separated by electrophoresis on a 2.5% Low-Melting agarose gel. A 160 bp fragment containing the modified hEGF gene was isolated from the gel.

 0.2 μg EcoRI-ClaI of the YEpGPD / SEC vector was ligated with 0.1 μg EcoRI-ClaI fragment of the modified hEGF gene and cells of the E. coli strain JM110 were transformed as described in Example 1. The obtained clones were analyzed for correct design using restriction analysis. Those clones that contained a unique BglII site (an internal site for the hEGF gene), and when cleaved with other restriction enzymes, formed characteristic fragments, as indicated in the table, corresponded to the correct design. The selected plasmid containing the hEGF gene of the YEpGPD / SEC vector was designated Yep40AB.

Example 3. Obtaining a strain of yeast 20B12 containing the plasmid Yep40AB
The cells of the recipient strain of S. cerevisiae 20B12 [a trpl-189 pep4-3] were transformed with YEpGPD / hEGF plasmid DNA according to the method of Ito et al. [13]. The yeast colony from the agar plate was transferred to 50 ml of liquid YEPD medium and incubated overnight at 30 ^° C. to an OD660 density of 1.0-2.0. The culture was centrifuged and the pellet was washed with a buffer of the following composition: 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA (TE buffer). The washed cells were resuspended in 50 ml of TE buffer containing 0.1 M LiAc and incubated for 2 hours at 30 ^° C. After centrifugation, the cells were suspended in 0.5 ml of LiAc / TE buffer, 10-20 μg of Yep40AB plasmid DNA was added and the mixture incubated for 30 min at 30 ^° C. Then 1 ml of 50% PEG4000 was added, mixed and incubated for 1 hour at 30 ^° C. Then heat shock followed for 5 minutes at 42 ^° C. The cells were centrifuged and washed with TE buffer. The washed cells were resuspended in 500 μl of water and plated 100 μl per Petri dish with selective agar (0.67% Yeast Nitrogen Base. Difco, 2% glucose, 2% agar). Transformants appeared after 2-4 days of incubation at 30 ^o C.

 Example 4. Testing hEFR in supernatans of the culture medium of recombinant strains.

To analyze the expression of hEFF derivatives in transformants of strain 20B12, clones 20B12 / YEpGPD / hEGF were grown in parallel, and as a negative control, strain 20B12 / YEpGPD / SEC in 1 ml of minimal YNB medium. After culturing for 12 hours at 30 ^° C., 0.5 ml of a suspension of cells from nine parallel cultures was transferred to 10 ml of YNB medium with 0.5% casamino acids and incubation continued for 48 hours. The supernatant of the culture medium was obtained after cell precipitation.

Determination of protein content with hEFF properties was carried out by enzyme-linked immunosorbent assay using poly - and monoclonal antibodies. 50 ng polyclonal rabbit anti-ChEFR rabbit antibodies were adsorbed onto 96-well immunological plates for 8 hours at 4 ^° C. All non-specific protein binding sites were blocked with 0.5% skimmed milk powder in 0.15 M NaCl buffer, 10 mM NaPi pH 7.4, 0.1% Tween 20 (PBS / Tween) for 30 min. Then the supernatants (in various dilutions) were transferred onto plates. Recombinant hEGF from Serva was used as a positive control. The plates were incubated for 2 hours at 37 ^° C. After repeated washing, anti-hEGF mAbs were added. After washing, plates with goat anti-mouse antibodies conjugated with horseradish peroxidase were incubated (dilution 1: 500, 2 hours at 37 ^° C. Bound peroxidase was detected by staining with 0.1 M citrate-sodium phosphate buffer pH 5.0, containing 0.16% orthophenylenediamine and 0.08% H ₂ O _2. After 15 minutes the reaction was stopped by adding 50 μl of 3M sulfuric acid and the absorbance was determined at a wavelength of 492 nm in a Multiscan instrument (Flow). Data obtained for various samples were averaged and compared with control samples hEFR company " Serva ". The level of hEFR expression thus determined in strain 20B12 / Yep40AB was 10 mg / L of culture medium at a density of 5 • 10 ⁸ cells / ml.

 Example 5. Isolation of hEFR from culture medium supernatants.

 The culture medium obtained after culturing the recombinant strain 20B12 / Yep40AB for 48 hours in a volume of 1 liter of YNB-CAA medium was centrifuged (12,000 rpm, 20 min) and the supernatant was applied to a column (2x4 cm) with Davisil C18 (90- 130 μm), balanced with 10% acetonitrile (2 volumes). The column was washed with 2 volumes of 20% acetonitrile with 0.1% TFA. Recombinant hEFR was eluted with 2 volumes of 60% acetonitrile containing 0.1% TFA at a flow rate of 5 ml / min. The eluate was lyophilized using Speed-Vac (Savant) and the final purification step was performed using reverse phase high performance liquid chromatography. For this, the drug after lyophilization was dissolved in 0.1% TFA, centrifuged to separate insoluble impurities, and applied to a Waters mBondapak C18 (0.78 x 30 cm) column. CHEFR was eluted using a linear gradient of acetonitrile concentration (0-60%) in 0.1% TFA / Fig. 3 /.

 According to SDS-electrophoresis in 15% SDS page, the purity of the resulting preparation was more than 98%. / Fig. 4/.

Literature
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 8. Morioka-Fujimoto K, et al. Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli. J Biol Chem. 1991 Jan 25; 266 (3): 1728-1732.

9. Yamagata H, Nakahama K., Suzuki Y, Kakinuma A, Tsukagoshi N, Udaka S Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc Natl Acad Sci USA 1989 May; 86 (10): 3589-3593
10. Urdea MS, Merryweather JP, Mullenbach GT, Coit D, Heberlein U, Valenzuela P, Barr PJ Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast. Proc Natl Acad Sci USA 1983 Dec; 80 (24): 7461-7465
11. Clare JJ, Romanes MA, Payment FB, Rowedder JE, Smith MA, Payne MM. Sreekrishna K, Henwood CA Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 1991 Sep 15; 105 (2): 205-212
12. Batchikova N.V., Ptitsyn L.R., Altman I.B., Stepanov A.I., Azhayev A.L. Chemical synthesis and cloning of the human epidermal growth factor gene Bioorgan. Chemistry, 1988 vol. 14, N 5 pp. 621-630.

 13. Ausubel F.M., Brent R.G., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K.A. Current Protocols in Molecular Biology. Massachusets General Hospital and Harvard Medical School, John Willey & Sons Inc., 1994.

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Claims (1)

 The yeast strain Saccharomyces cerevisial 20 B 12 containing the recombinant plasmid DNA YEp 40 AB, VKM CR-349D, is a producer of human epidermal growth factor.

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