EA025626B1 - PRODUCER OF HUMAN EPIDERMAL GROWTH FACTOR (rhEGF) - Google Patents

Abstract

The invention is related to a method for production of recombinant human epidermal growth factor by cultivating a new rhEGF-producing E.coli strain which was obtained by transformation by a recombinant plasmid containing a hEGF gene included in the expression cassette by the electroporation method. The producer obtained is cultivated in selective conditions, rhEGF synthesis is induced by means of an inductor, namely, isopropylthiogalactoside (IPTG). The strain is able to produce rhEGF in an amount of up to 200 mg per litre of culture. After cultivation, the cells are collected by centrifugation, lysed, and inclusion bodies are isolated. The rhEGF isolation procedure includes isolation of inclusion bodies, EGF renaturation, acidic sedimentation of ballast proteins and chromatographic purification on a strong cation exchanger. Electophoretically pure EGF is produced with a yield of up to 90 mg from a litre of culture. The recombinant hEGF corresponds to natural hEGF in its biological, physical and chemical properties. The invention allows production of biologically active rhEGF with a high yield.

Description

The invention relates to biotechnology, in particular to a technology for producing recombinant proteins, specifically to producing recombinant human epidermal growth factor (rhEFR) and can be used in pharmacologists and medicine to create new anti-burn and wound healing agents, for the diagnosis of malignant diseases associated with hyperproduction of the hEFR receptor , as well as in the creation of new methods for targeted delivery of certain therapeutic agents to target cells specifically expressing the chEF receptor . The resulting recombinant rhEFR preparations can also be used for cosmetology purposes, as well as for scientific research, including as components of culture media for animal cell cultures.

BACKGROUND OF THE INVENTION rhEFR is a peptide hormone produced by a variety of tissues and found in almost all body fluids. EGF is synthesized in the form of a precursor with a length of 1217 amino acids and is subjected to further proteolytic processing with the formation of a mature protein with a characteristic size of 53 amino acid residues and with three internal disulfide bonds [1].

EGF acts on cells containing the corresponding receptor on its surface, mainly epithelial and connective tissues, and stimulates their growth and division.

Biologically active hEFR in preparative amounts was originally obtained from human urine [2,3]. But the low content of the target product and the lack of sources of raw materials logically led to the need for the expression of this protein in bacterial cell cultures using recombinant DNA technology [4,5]. However, the expression of hEFR in bacterial cells is limited due to the high toxicity of the target product for the microbial host cell.

There are two methods to overcome this limitation:

1) cloning of the gene of the precursor protein in the expression plasmid, followed by cleavage of the signal peptide and secretion of the mature protein into the extracellular space;

2) the transition of the target protein into an insoluble form and its localization in the cell in the form of inclusion bodies.

Currently, the practice primarily uses the first option. Recombinant plasmids are known that control the synthesis and secretion of rhEFR in bacteria cultures Ε.οοΙί [6,7,8] with a maximum productivity of up to 70 mg per liter of culture, as well as various types of VasShik [9, 10], Rkeibiotoyak [11] and yeast [ 12,13,14,15] with a slightly higher yield of the target product. However, this approach is not economically justified, because due to the relatively low content of the target product in the microbial culture, it is necessary to process large volumes of raw materials, which significantly complicates the technology for isolation and purification of rhEFR. In the case of VasShik and Rkeibotoyak bacteria, an additional limitation is the environmental hazard of the use of sporulating microorganisms in the production. The second drawback is the partial proteolysis of the product during its accumulation in a soluble form in the culture fluid, which is accessible for the action of proteases secreted by the cells of microorganisms as a result of secretion or lysis of part of the cells in the population [16, 17]. This leads to heterogeneity of the recombinant protein and a decrease in the yield of the target product.

Thus, RF patent 2150551 is known [14], in which a recombinant yeast strain 8.seteu1K1ae is claimed, from the culture medium of which up to 10 mg / l of native rhEFR was isolated.

Also known is a recombinant strain of E.coP. which is able to secrete rhEFR into the culture fluid [6], from which the target product was isolated by immunoaffinity chromatography with a yield of 1.2 mg from 1 liter of culture. This amounted to about 95% of its content in the original culture [18].

The second approach is to design producer strains in which rEFR forms inclusion bodies that protect the target product from proteolysis. However, a drawback of this approach is the mechanism of negative autoselection in cultures of such strains, which is due to the presence of a certain level of spontaneous synthesis of rhEFR and its toxicity to the host cell. This phenomenon leads to a rapid decrease in the productivity of the obtained producer strains during their cultivation in comparison with the primary transformed bacterial culture and, as a result, to a low yield of the target protein.

In most known works, the authors circumvent this obstacle by synthesizing a larger recombinant protein (chimera), in which the amino acid sequence of EGF is connected to the sequence of another protein [19, 20, 21]. A variant of this approach is the extension of the EGF sequence by 6 or more histidine residues (NkChaddeb) [22, 23]. This approach gives good results in laboratory conditions, but it is not technologically advanced, since in order to obtain the target product, it is necessary to carry out an additional expensive and difficult to reproduce under production conditions stage of enzymatic cleavage of the additional peptide.

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The prototype of the invention is a method for obtaining authentic human EGF by constructing a recombinant plasmid based on the vector pET21b with a low level of spontaneous synthesis in E. soybean, which reduces the level of negative auto-selection [24]. Subsequent isolation of inclusion bodies, their dissolution, renaturation and purification by HPLC chromatography provide biologically active EGF with a yield of up to 10 mg per liter of the original culture.

Disclosure of invention

The inventive objective of the invention is to obtain a highly purified biologically active fully mature recombinant rhEFR in high yield.

For this, a recombinant plasmid ρΜΖΕΗΕΟΡ was constructed, transformation with this plasmid E. soy was performed to obtain a stable E. soy strain producing rhEFR, E. soy B21 / OE3 ρΜΖΕΗΕΟΡ, in which spontaneous synthesis of rhEFR was practically excluded and, thus, the mechanism was blocked negative autoselection (which allowed to maintain a high level of rhEFR synthesis throughout the entire fermentation cycle), methods for cultivating the obtained strain on selective nutrient media followed by isolation of rhEFR from Taurus on teaching, its renaturation and purification.

The obtained strain I.soP was deposited in the All-Russian collection of industrial microorganisms under the number VKPM: B-11670.

Thus, the claimed producer strain of human epidermal growth factor (rhEFR) ΒΕ21 / ΟΕ3 ρΜΖΕΗΕΟΡ, (VKPM No. B11670) containing the plasmid DNA ρΜΖΕΗΕΟΡ with the map corresponding to the drawing and containing the expression cassette, with a nucleotide sequence of 8ΕΟ GO No. 1.

The strain is preferably characterized by an rhEFF production level of up to 200 mg / L culture.

A method for producing the producer strain described above is also described by transforming the Ε. ^ Ϊ́ί ΒΕ21 / ΟΕ3 cells with the ρΜΖΕΗΕΟΡ plasmid, which contains the semisynthetic mature hEFR gene encoding an authentic rhEFR protein inserted into the expression plasmid cassette under the control of the transcription promoter and transcriptional terminator subsequent selection.

A method for producing rhEFR is also claimed, including culturing cells of the strain I.coP ΒΕ21 / ΟΕ3 ρΜΖΕΗΕΟΡ under selective conditions, inducing synthesis of rhEFR, separating cells from the culture fluid, lysing them, isolating and dissolving inclusion bodies containing rf EGF, its renaturation, acid deposition of ballast proteins and chromatographic purification of rhEFR on cation exchange resin.

The method for producing rhEFFR is preferably characterized in that the cultivation of the B.coP strain of the rhEFF producer - ΒΕ21 / ΏΕ3 ρΜΖΕΗΕΟΡ is carried out in a gas-vortex bioreactor in a selective medium at 35-37 ° C, preferably at 37 ° C, the turbine rotation speed is up to 1200-2000 rpm / min, automatic control of pH and pO ₂ during aeration with air.

The synthesis of rhEFF is induced by the addition of a solution of isopropylthiogalactoside (IPTG) and fermentation is carried out to OE _{600 of the} culture 6.0 ± 0.5, the cells are separated from the culture fluid from the biomass by centrifugation.

The separation and concentration of cells from biomass is carried out by diafiltration on a membrane with a pore diameter of 0.45 μm.

Cell lysis can be carried out by enzymatically sequential treatment of the cell suspension with lysozyme, freezing, thawing and deoxyribonuclease.

Cell lysis can be carried out by treating the suspension in an ultrasonic or mechanical disintegrator.

Isolation of inclusion bodies is carried out by differential centrifugation or diafiltration on a module with a pore diameter of 0.2-0.45 microns.

Dissolution of inclusion bodies is carried out in the presence of guanidine chloride and sulfhydryl compounds. Dissolution of inclusion bodies can also be carried out in the presence of urea and sulfhydryl compounds.

The renaturation of rhEFR is expediently carried out by dilution 20-100 times in an acetate buffer solution and prolonged incubation with stirring in the cold.

Isolation of rhEFF is preferably carried out by acid deposition of ballast proteins by lowering the pH with acetic acid and centrifuging or filtering.

The rHEFR is purified by chromatography on a column packed with strong cation exchange resin, for example on a column packed with δ-Sepharose EP.

The elution of rhEFR is preferably carried out with an acetate buffer containing Tween-20, and the pH of the eluate is adjusted to 7.1 ± 0.1 by the addition of, for example, sodium hydroxide.

The content of the target rhEFR in the preparation exceeded 98%, and the specific activity of the obtained rhEFR was not lower than 1.0 x 10 ⁶ IU / mg protein.

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Brief Description of the Drawings

The drawing shows a genetic map of the expression plasmid ρΜΖΕΙιΕΟΕ. It contains T7 rgot — the promoter of the bacteriophage T7, ΕΟΡ — the hEFF gene, T7 1egt — the terminator for the transcription of the bacteriophage T7, K — the antibiotic resistance gene, for example, the gene for resistance to ampicillin, kanamycin, etc., and Οτι — the origin of the replication of the pVK322 vector.

The implementation of the invention

The obtained advantages of the developed method are illustrated by examples.

Example 1. Obtaining a recombinant plasmid ρΜΖΕΗΕΟΡ.

Plasmid ρΜΖΕΗΕΟΡ is a vector containing a semisynthetic gene for mature rhEFR.

The semi-synthetic rhEFR gene was generated by PCR on a DNA matrix obtained by the chemical synthesis of oligonucleotides followed by ligase assembly. In the reaction used oligonucleotide primers:

containing initiating and terminating codons, as well as restriction sites necessary for cloning. At the end of the reaction, the product was treated sequentially with WatH1 endonuclease, T4 bacteriophage DNA polymerase and ΝάβΙ endonuclease. The final product was purified from excess salts and by-products of the reaction at Sephadex 0-50. As a result, a 170 bp DNA fragment was obtained.

The DNA of the vector was linearized by treatment with restriction endonuclease Βρί11102Ι, then it was sequentially treated with bacteriophage T4 DNA polymerase and онβΙ endonuclease.

A 170 bp fragment containing the mature rhEFR gene was cloned into the obtained linear vector form in the ligation reaction of the T4 bacteriophage DNA ligase. The cells of the strain Ε.αοΐί ΌΗ10Β (0FcKBK, genotype P ^- tag A, D (tg-b5bKM§-tag BBC), φ806ΕκΖΔΜ 15, D1aeX74, beoK, gesA1, eibA1, aaO139, Δ1α69. , yes 1K / _. τρδΕ, ηυρΟ). Transformed cells were plated on Petri dishes with agarized LB medium containing 100 μg / ml ampicillin. Plasmid DNAs were isolated from the obtained colonies and screened by PCR and gel electrophoresis followed by sequencing. As a result, a recombinant clone Ε ^ οΐί IN10B containing the desired plasmid ρΜΖΕΙιΕΟΕ was obtained. Plasmid DNA ρΜΖΕΙιΕΟΕ was isolated from the overnight culture of B. soybean cells ONYUV / ρΜΖΕΙιΕΟΕ by a method based on lysis of biomass with alkali followed by purification.

Example 2. Characterization of a plasmid that controls the synthesis of rhEFR.

The resulting plasmid ρΜΖΕΙιΕΟΕ (see the drawing), has a size of 4.7 thousand base pairs (bp), contains a mature hEFR gene encoding an authentic rhEFR protein under the control of the promoter and terminator of phage T7 (see Nucleotide sequence of BCC GO No. 1 )

Plasmid ρΜΖΕΙιΕΟΕ consists of the following DNA fragments: a DNA fragment with a 170 bp mature hEFR gene generated by polymerase chain reaction (PCR) on a DNA matrix obtained by the chemical synthesis of oligonucleotides followed by ligase assembly, with oligonucleotide seeds

sequentially treated with WatH1 endonuclease, T4 bacteriophage DNA polymerase and She1 endonuclease;

vector plasmid DNA fragment obtained by sequential treatment with Bree 11021 endonuclease, T4 bacteriophage DNA polymerase and She1 endonuclease.

Example 3. Obtaining a cell culture B. coN - producer of rhEFR.

B. sON cells VB21GOB3 (Rgoteda, genotype Ε-, otρΤ, NkbZV (g _B - g _B -), bst, da1, [IB3]) were transformed with plasmid ρΜΖΕΗΕΟΕ by electroporation.

Transformed cells were plated on Petri dishes with agarized BV medium containing 100 μg / ml ampicillin and incubated for 14–18 h at 37 ° C. At the end of the incubation, the biological material of a separate colony was inoculated with 2 ml of BW liquid medium containing 100 μg / ml ampicillin, cultivated for 3 hours at 37 ° C with stirring, isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM, and cultivation continued for 4 hours. At the end culturing cells were collected by centrifugation, lysed in a solution containing 0.125 M Tris-HCl, pH 6.8, 10% P-mercaptoethanol and 4% δΌδ by boiling for 5 min. The amount of rhEFF protein in the lysate was determined by enzyme-linked immunosorbent assay (ELISA). As a result, a clone Β ^ 21 / ^ B3 ρΜΖΕΙιΕΟΕ was selected, providing a level of expression of rhEFR up to 200 mg per liter of culture. The strain Ε. ^ 1 ί the producer of rhEFR was deposited in the bacterial museum of the Protein circuit LLC under the number: Β ^ 21 / ^ B3 ρΜΖΕΙιΕΟΕ and in the VKPM under the number: B-11670.

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Example 4. Fermentation of a producer strain of rhEFR.

A material of 3-5 separately growing colonies was seeded with 50 ml of LB medium containing 100 μg / ml ampicillin into 0.5 L flasks and cultured for 16-18 hours at 37 ° C on a thermostated shaker. The resulting material was plated in a ratio of 1:20 (V / C) in 2 L flasks containing 200 ml of LB medium with 100 μg / ml ampicillin. The flasks were incubated for 3-6 hours at a temperature of 37 ° C on a thermostated shaker until an optical density of OE ₆₀₀ = 1.5-2.0 was reached. Dimethyl sulfoxide was added to the resulting cell culture to a final concentration of 10% (V / V), 50 ml were aliquoted and frozen in a refrigerator at -70 ° C. The resulting cell culture (seed) can be stored at -70 ° C for at least 4 months. or at -150 ° C - up to 2 years.

A cryopreserved cell culture (seed) was introduced in an amount of 350 ml into the BIOK gas-vortex bioreactor (ZAO Sayany) containing 7 l of fresh LB medium containing 100 μg / ml ampicillin.

The fermenter was connected to a compressed air supply line from a compressor with a sterilizing filter. The fermenter heating was turned on, automatic temperature control was set to 37 ° C and the turbine rotation speed was adjusted to 1200-1500 rpm. The pH of the medium in the fermenter was controlled: keeping the pH value no higher than 7.6.

The air pressure on the compressor was set at (0.20 ± 0.05) atm. The pO ₂ value was controlled; after the pO _{2 was} reduced to a value of (15 ± 5), the compressed air supply to the barbator was gradually switched on, maintaining the pO ₂ value within (20 ± 10). After a complete switch to the bubbler, the pO ₂ value was maintained, gradually increasing the pressure of the compressed air and controlling the air flow using a rotameter. Air flow should remain within (10-15) l / min.

After 2 hours of cultivation at intervals of 30 minutes, culture samples were taken to determine the optical density. After reaching the optical density of the culture value of OE ₆₀₀ equal to 1.0-1.5 in the capacity of the fermenter served sterile inducer solution (IPTG) to a final concentration of 1 mm. After induction, the pO ₂ value was increased to values> 30 and maintained at this level until the end of fermentation, increasing the pressure on the compressor and the mixing speed.

After reaching a pressure value in the fermenter of 1.8-2.0 atm, the required pO ₂ value in the culture was maintained by pulsing supplying oxygen from the cylinder through the gearbox to the compressed air supply system. This made it possible to maintain cultivation parameters: pressure in the range of 1.8-2.0 atm, stirring speed of 2000-2300 rpm. The pH of the medium was maintained at a level of (7.6-7.8) until the end of fermentation.

2 hours after the addition of the inducer and at intervals of 30 minutes, culture samples were taken. The optical density of the culture and the morphology of the cells were monitored under a microscope. The dynamics of an increase in optical density and the formation of inclusion bodies were observed. 5-6 hours after induction, the optical density reached a plateau at the level of OE600 equal to (6.0 ± 0.5). Almost all cells contain inclusion bodies, 1-3 per cell, from small dark inclusions to black bodies, which are 2-3 times larger than the thickness of the cell.

The optimal level of rhEFR output in the analyzed culture samples was achieved 5 hours after the addition of the inducer; with an increase in the cultivation duration of more than 5.5 hours after the addition of the inductor, a decrease in the rhEFR yield was observed.

The average biomass yield during cultivation under optimal conditions was 7.2 g / l with an rhEFR content of 27.2 mg / g cells.

Example 5. Isolation of inclusion bodies.

A) Differential centrifugation method.

An equal volume of biomass lysis solution was added to the frozen pellet of 8 g of cells: 10 mM Tris-HCl, 10 mM EDTA, pH 7.4, 80 μg / ml ΡΜδΡ. The biomass was thawed, suspended on a Vortex, a weighed sample of lysozyme was added with stirring at the rate of 1 mg / g of cells, diluted twice with a solution for lysis of biomass and frozen. Then, the suspension was thawed in a water bath at room temperature, left under stirring for 20 min at room temperature, a solution of deoxyribonuclease up to 10 μg / ml and a weighed portion of magnesium sulfate were added to a final concentration of 11-12 mm. The suspension was processed in a blender for 5-7 minutes, after which it was diluted 10 times with a solution for lysis of biomass.

The resulting bacterial cell lysate was clarified by centrifugation at 9000 rpm, 20 min (centrifuge 42-21, Beckman, USA, rotor 1L-14).

The precipitate after centrifugation was successively washed with the following solutions, each time suspending the precipitate and precipitating by centrifugation under the conditions given above:

mM Tris-HC1, pH 8.0, 1 mM EDTA, 0.1% Triton X-100, 1 L;

mM Tris-HC1, pH 8.0, 1 mM EDTA, 0.05% tween-20.1 L;

mM Tris-HC1, pH 8.0, 1 mM EDTA, 200 ml; isopropyl alcohol, cooled to -20 ° C, 100 ml; deionized water, 0.7 l.

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The precipitate was weighed and suspended in an equal volume of deionized water. A 50% suspension was stored at -20 ° C. 1.73 g of inclusion bodies were obtained (22% of the biomass weight). The yield of purified EGF was about 25.4 mg per g of inclusion bodies.

B) Diafiltration on the module νίν; · ιΠο \ ν 200, νίν; · ιΠο \ ν Yb. Great Britain with 0 pores 0.2 microns. Lysis 16.2 g of biomass was produced as described above (Example 5A). The clarified lysate was adjusted to 800 ml with a lysis solution and concentrated on a microfiltration module ννναΠον 200 with a membrane with a pore diameter of 0.2 μm with a Mayegyeh b / δ peristaltic pump at an operating pressure of 1.5 atm. The volume of the suspension was adjusted to 200 ml. The suspension was sequentially washed in diafiltration mode with the following solutions:

mM Tris-HC1, pH 8.0, 1 mM EDTA, 0.1% Triton X-100, 400 ml, 100-150 ml are added, concentrated to 200 ml;

mM Tris-HC1, pH 8.0, 1 mM EDTA, 0.05% tween-20, 200 ml, 50-70 ml are added, concentrated to 25 ml;

mm Tris-HC1, pH 8.0, 1 mm EDTA, 50 ml, add 15-20 ml, concentrate to 25 ml; isopropyl alcohol, cooled to -20 ° C, 50 ml, add 15-20 ml, concentrate to ml;

deionized water, 75 ml, add 15-20 ml, concentrate to 25 ml.

Using a pump, the suspension was transferred to a centrifuge cup, the module was washed with deionized water (3 times 20 ml). The suspension and washings were combined, inclusion bodies were collected by centrifugation at 12,000 rpm at 4 ° C for 30 minutes, the centrifuge 62-21. Beckman, USA, rotor 1Λ14. The supernatant was drained, the precipitate was weighed and suspended in an equal volume of deionized water. A 50% suspension was stored at -20 ° C.

Received 1.8 g of inclusion bodies (11.1% of the weight of the biomass) with an rhEFR content of 116 mg per 1 g of inclusion bodies.

C) Diafiltration on a Myrog module with a Re1Bkoy membrane, 0 pore 0.45 μm.

Lysis 86.6 g of biomass was produced as described above (Example 5A). The clarified lysate was adjusted to 4 L with a solution for cell lysis and concentrated on a MPProg microfiltration module with a Re1Bkoy membrane, 0 pore 0.45 μm to 800 ml.

The suspension was sequentially washed in diafiltration mode with the following solutions:

mm Tris-HC1, pH 8.0, 1 mm EDTA, 0.1% Triton X-100, 2 L, 200-250 ml are added, concentrated to 800 ml;

mM Tris-HC1, pH 8.0, 1 mM EDTA, 0.05% tween-20, 1 L, 100-150 ml are added, concentrated to 100 ml;

mM Tris-HC1, pH 8.0, 1 mM EDTA, 250 ml, 40-50 ml are added, concentrated to 100 ml; isopropyl alcohol, cooled to -20 ° C, 250 ml, 40-50 ml are added, concentrated to

100 ml;

deionized water, 300 ml, 40-50 ml are added, concentrated to 100 ml.

Using a pump, the suspension was transferred to a centrifuge cup, the module was washed with deionized water (3 times 50 ml). The suspension and washings were combined, inclusion bodies were collected by centrifugation at 12,000 rpm at 4 ° C for 30 minutes, a centrifuge 12-21, Beckman, USA, rotor 1Λ14. The supernatant was drained, the precipitate was weighed and suspended in an equal volume of deionized water. A 50% suspension was stored at -20 ° C.

Received 16.1 g inclusion Taurus (18.6% of the weight of the biomass) with an rhEFR content of 155 mg per 1 g inclusion Taurus.

Example 6. Dissolution of inclusion bodies, renaturation and purification of rhEFR.

32.2 g of a 50% aqueous suspension of TB were thawed at room temperature and guanidine chloride was added with stirring on a magnetic stirrer to a final concentration of 7 M and 2-mercaptoethanol to a final concentration of 100 mM. The pH of the solution was adjusted to (8.0 ± 0.1) by adding dropwise 0.5 M sodium hydroxide. Incubated with stirring for 2 hours at room temperature.

In a container with a capacity of 10 l, located in a refrigerator, 5 l of 50 mM acetate buffer pH 8.0, pre-cooled to a temperature of 5 ° C, was placed. With constant stirring, a solution of inclusion bodies was added in 5 ml portions. They were incubated with stirring for 24 hours. At the end of the incubation, the solution was acidified to pH 3.0 by adding glacial acetic acid dropwise. The solution was clarified by centrifugation at 9000 rpm at 4 ° C for 30 minutes.

The supernatant was applied to a K 25x20 column with a δ-Sepharose PP sorbent at a rate of 5 ml / min, the column was washed from unsorbed material, passing 300 ml of 50 mM acetate buffer pH 3.0. EGF was eluted with 50 mM acetate buffer, pH 5.0, containing 0.05% Tween-20 (ν / ν). The eluate was collected based on the readings of the absorptiometer. The pH of the eluate was immediately adjusted to 7.1 ± 0.1 by the addition of 0.5 M sodium hydroxide.

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Received 140 ml of rhEFR solution with a concentration of 9.2 mg / ml. According to δΌδ-electrophoresis in PAGE, the purity of the obtained preparation exceeded 98%

Example 7. Determination of the biological activity of rhEFR.

The solution of the studied rhEFF with a concentration of 9.2 mg / ml was sterilely diluted 9.2 times in GOMEM medium (ΙΟΝ, USA) to a concentration of 1 mg / ml.

150 μl of GOMEM medium was added to the wells of a 96-well flat-bottomed Co81ag plate. Then, in the wells of the left half (6 holes out of 12 in each row), the plates were sequentially diluted in the GOMEM medium with the 1st international standard for EGF (1 81 1p1etpayopa1 81aiyat) - from the National Institute of Biological Standards and Control (ΝΙΒδΟ), Great Britain (hess ΌΝΑ, Lishap 1ure, sobe 91/530). For this, 4.8 units of activity in 150 μl of standard EGF were added to six wells of the left half of the first row (row A) with a multichannel pipette, mixed by pipetting, 150 μl were transferred to the wells of the left half of the next row (row B), and the transfer procedure was repeated and stirring up to and including the sixth row of the tablet (row P), from which 150 μl of the solution was discarded. 150 μl of rhEFR solution with a concentration of 1 mg / ml were added to six wells of the right half of the first row (row A) with a multichannel pipette. Then, the test sample rhEFR was successively diluted in the wells of the right half of the plate to the last (row H), in the same way as was described for standard EGF. To all wells of the plate were added 150 μl of a suspension of HC 11 cell line (RpebpsN M1e8set 1p8Y1i1e, Basel, Switzerland) at 5 thousand per well in GOMEM medium (ΙΗΝ, USA) containing 20% bovine fetal serum (Nuclope, USA) . Thus, the final activity of standard EGF in rows AR for the wells of the left half of the tablet was 8 IU / ml (row A), 4 IU / ml (row B), 2 IU / ml (row C), 1 IU / ml ( row Ό), 0.5 IU / ml (row E) and 0.25 IU / ml (row P). In the wells of the right half of the plate, the sample of the studied rhEFR was sequentially diluted in 18 (row A), 36 (row B), 72 (row C), 144 (row Ό), 288 (row E), 576 (row P), 1152 ( row O) and 2304 times (row H).

After 4 days of incubation of the tablet in a CO ₂ incubator at 37 ° C, the wells of the tablet were washed twice with physiological saline, 40 μl of a solution containing 20% methyl alcohol and 20 mg / ml crystal violet were added, and incubated for 20 min at room temperature. The wells of the plate were washed seven times with tap water and 100 μl of a solution containing 5% glycerol and 20 mg / ml sodium dodecyl sulfate was added. Light absorption was determined for each well at a wavelength of 540 nm on an ΒΙΟ-ΚΑΌ reader, tobe 3550; the average of six values of one dilution was calculated. From the average values, the average for the wells of the left half of the O and H rows was subtracted (negative control of cell proliferation without adding EGF). A calibration curve was constructed for a standard EGF sample and a curve showing the proliferative effect of the studied rhEFR sample on its dilution. Using the linear portion of the calibration curve, the biological activity of the test rhEFR sample was determined, which was 9.2 x 10 ⁶ IU / ml.

The specific activity (UA) in IU per mg of protein was calculated by the formula: UA = BA / SB, where BA is the biological activity in IU / ml, SB is the protein concentration in mg / ml. The specific activity of the obtained rhEFF was 1.0 x 10 ⁶ IU / mg protein.

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Literature

1. Century Ο.Ι., Ροη§ Ν.Μ., Κίβιηρίβη M.M. e1 a1. Nitap erIegta! §GOM Gasyug Przesigzog: ίϋΝΑ zeyeieppse, exprzyzyup ίη νίίτο apb depeg ^ atgabop.// 14is1eyu As1bz Kez, -1986, U.14.-P.8427-844b.

2. Pat. LR No. 58099418.1983, class A61K35 / 22.

3. Pat. LR No. 62030718.1987, class A61K35 / 22.

4. Pat. LR No. 62040290.1987, class S07H21 / 04.

5. Batchikova N.V., Skaptsova N.V., Tvardovskaya S.E. et al. Chemical synthesis and cloning of the human epidermal growth factor gene. // Bioorgan, chemistry. - 1988.V.14 (5) .- C .621-630.

6. Ptitsyn L.R., Altman I.B. Extracellular production of recombinant human epidermal growth factor (LEPO) in the cells of Euxepius soybean. // Bioorgan. Chemistry.-1999.-T.25 (12) .- S. 923-929.

7. Pat. SI No. 1279288, 2001, class A61K38 / 18.

8. Pat. ER No. 0352839, 1990, cl. S07K14 / 485.

9. Pat. LR No. 2031682, 1990, class S07K 14/32.

10. \ Wap§ V., Wagsch X., \ Wu K., Ηί§Η-Ιβνβ1 prgbisbop Г 1 из из из Ю Ю Ю ί ί а а а Вас Вас Вас Вас Вас Вас Вас V .4 .4 .4 .4 .4 .4 .4 .4 .4. 3), - R.223-231.

11. Nauave N., SYuata A., number \ Chapo M Zesgebop og itap er1begta1 ^ τον, ΐΗ GasYug (EOP) ίη aiYu1gorYs shikigami Ly and gesottat ubgo§ep- ibIyp £ asYupshp, Rveibotopaz rzeiboYaaua, saggut§ goab-ovT- hapde EOR zsgebop chesYug pK8EOP2. // ΐρρΐ Εηνίτοη M1sgoLu1.-1994.-U.60 (9) P.3336-3342.

12. Pat. from No. 5102789,1992, class S07K14 / 485.

13. Pat. KK No. 920008372, 1992, class С12Ы15 / 12.

14. Pat. RF №2150501, 2000, κπ.Ο12Ν1 / 19.

15. Pat. ΨΟ No.2012060666,2012, class S07K14 / 485.

16. C1age L.L., Kotapoz M.A., Kautep! RV, e! A1, Rgobisbop OG Toise Eriegta! § Goal Gasuгn ίη uav1: Ηίβΐι-ΐβνβΐ zesgebop from § Ы Ы раз раз раз з в в!!! В со Ш Ш т д ти ти ти сеп сеп // // // // // //----.

17. Natsa Ρ.Ύ., Kasko R., Cialo V.V. Rgobisbop apb zesgebop OGu1o £ 1s11u asbuye Litap er1begta1 §gomb GasUg ίη Uaggoila Nro1ubsa. // Sip ^- OepeS- 1998.- U.Z. - No. 3.- R.231-7.

18. Pat. RF №2108343, 1998, class S07K14 / 485.

19. Pat. KK No. 930001386, 1993, class С12Ы15 / 19

20. Pat. ΟΝ No. 1765929, 2006, class A61K38 / 18.

21. Pat. ΟΝ No. 101875699, 2010.cl.A61K38 / 18.

22. Gay .Τ.Υ., Υοοη P.8, SNipd Ι.Υ. e1 a1. 8sa1e-ir rgosezog Gog exrgeziup apb hepaYugabop οί hesotypap! Hitap Ergbegt! §GOM GASUGGGOT EXCLUSIVE SOCIETY SHOULDER.// VYUYUSPO1 Arr1 Vyuset.-2000.-U.31 (P13) .- P.245-248.

23. Zagta K., Baib R.U.S., Zaz | bNag P e1 a1. KesotTap! Ап ап ап 1 1 1 1 1 β β Γ Ю Ю Ю 2008 2008 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7 .7. .

24. Sölég E.R., Sebapo L, () and its 1 E, be Pogepz K .. Cloptd, exprunzip apb ripisbop dlbnrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrr. Vyubeb 1lGe 8sG-2003.-U.788 (1) .- P.113-123.

- 7 025626

List of sequences <110> Proteinovy Kontur LLC, LLC ΡΕΟΐβϊηονίγ KopRig <120> Producer of human epidermal growth factor (rhEFR) <130> Producer of human epidermal growth factor (rhEFR) <160> 1 <170> RaRepRSh νβΓΒΪοη 3.5 <210> <211> 328 <212> DNA <213> Artificial sequence <220>

<223> The nucleotide sequence of the expression cassette of the ρΜΖΕΡΕΟΡ plasmid containing the sequences of the promoter of the T7 bacteriophage (T7 rgototoReg), the ribosome binding site (CVZ), the ECG gene, and the T7 bacteriophage transcription terminator (T7 RegshZpRog)

<400> 1 Ada £ c1: Ada £ sssdsdaaaR RaaRasdasR sasRaRaddd adassasas ddRRRssssRs 60 Baaaaaaaaa ΡΉΐ: ds; -aaas RRRaadadd ADARARASAR ARDAARRSRD asRsRdaaRd 120 sssaEdE sasdasddrr asRdsRRds sdasddrdrr rdsardras RsdaadsRsR 180 ddasaaaas dsRRdsaaRR dsdrrdrrdd RRasRsddR daasdrrdss aaRassdada 240 ^ c ^ daaa ^ dd RdddaasRds drrdradradd ARDSDSARAAA sRadsaRaas sssrrdddds 300 c £ c £ aaasdd DRRSRRddadd drrrrrrd 328

CLAIM

Claims (22)

1. The strain producing human epidermal factor (rhEFR) ΒΕ21 / ΏΕ3 ρΜΖΕΕΕΟΡ, (VKPM No. B11670) containing plasmid DNA ρΜΖΕΗΕΟΡ with a map corresponding to the drawing and containing an expression cassette with a nucleotide sequence corresponding to 8Ε0 GO No. 1. 2. The strain according to claim 1, characterized by the level of production of rhEFR up to 200 mg / l of culture. 3. A method for producing a producer strain, characterized by claims 1, 2, by transforming E.coN VB21GOE3 cells with the ρΜΖΕΙιΕΟΕ plasmid, which contains the semisynthetic mature hEFR gene encoding an authentic rhEFR protein inserted into the expression plasmid cassette under the control of the promoter and transcription terminator T7 followed by selection. 4. A method for producing rhEFR, including culturing cells of the strain Εχοΐί ΒΕ21 / ΏΕ3 ρΜΖΕΙιΕΟΕ, characterized in claims 1, 2, under selective conditions, inducing synthesis of rhEFR, separating cells from the culture fluid, their lysis, isolation and dissolution of inclusion bodies containing rhEFR, its renaturation, acid deposition of ballast proteins, and chromatographic purification of rhEFR on cation exchange resin. 5. The method according to claim 4, characterized in that the cultivation of strain Ε + ο1ί of the producer of rhEFR ΒΕ21 / ΌΕ3 ρΜΖΕΙιΕΟΕ - is carried out in a gas-vortex bioreactor in a selective medium at 37 ° C, the turbine rotation speed is up to 1200-2000 rpm, automatic pH control and pO ₂ during aeration with air. 6. The method according to claim 4, characterized in that the synthesis of rhEFR is induced by adding a solution of isopropylthiogalactoside (IPTG) and fermentation is carried out to OE ₆₀₀ culture 6.0 ± 0.5. 7. The method according to claim 4, characterized in that the separation of cells from the culture fluid from the biomass is carried out by centrifugation. 8. The method according to claim 4, characterized in that the separation and concentration of cells from the biomass is carried out by diafiltration on a membrane with a pore diameter of 0.45 μm. 9. The method according to claim 4, characterized in that the lysis of the cells is carried out by enzymatically sequential treatment of the cell suspension with lysozyme, freezing, thawing and deoxyribonucleotase. 10. The method according to claim 4, characterized in that the lysis of the cells is carried out by processing the suspension in an ultrasonic disintegrator. 11. The method according to claim 4, characterized in that the lysis of the cells is carried out by processing the suspension in a mechanical disintegrator. 12. The method according to claim 4, characterized in that the selection of inclusion bodies is carried out by differential centrifugation. 13. The method according to claim 4, characterized in that the selection of inclusion bodies is carried out by diafiltration on a module with a pore diameter of 0.2 μm. 14. The method according to claim 4, characterized in that the selection of inclusion bodies is carried out by diafiltration on a module with a pore diameter of 0.45 μm. 15. The method according to claim 4, characterized in that the dissolution of the inclusion bodies is carried out in the presence of guanidine chloride and sulfhydryl compounds. 16. The method according to claim 4, characterized in that the dissolution of the inclusion bodies is carried out in the presence of urea and sulfhydryl compounds. 17. The method according to claim 4, characterized in that the renaturation of rhEFR is carried out by dilution in 20-100 times in a solution of acetate buffer and prolonged incubation with stirring in the cold. 18. The method according to claim 4, characterized in that the isolation of rhEFF is carried out by acid deposition of ballast proteins by lowering the pH with acetic acid and centrifuging or filtering. 19. The method according to claim 4, characterized in that the rHEFR is purified by column chromatography packed with strong cation exchanger. 20. The method according to claim 4, characterized in that the purification of rhEFR is carried out by chromatography on a column packed with δ-Sepharose PP. 21. The method according to claim 4, characterized in that the elution of rhEFR is carried out with an acetate buffer containing Tween-20, and the pH of the eluate is immediately adjusted to 7.1 ± 0.1 by adding sodium hydroxide. 22. The method according to claim 4, characterized in that as a result of purification of rhEFR its content in the preparation exceeded 98% and the specific activity of the obtained rhEFR was not lower than 1.0 x 10 ⁶ IU / mg protein. Producer of human epidermal growth factor (rhEFR). Genetic map of the expression plasmid ρΜΖΕΕΕΟΡ. T7 rgot is the promoter of the bacteriophage T7, BOR is the hEFF gene, T7 1cgt is the terminator for the transcription of the bacteriophage T7, K is the antibiotic resistance gene, for example, the gene for resistance to ampicillin, kanamycin, etc.