Method of isolating and purifying recombinant human growth hormone secreted by saccharomyces cerevisiae yeast

FIELD: chemistry.

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

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

8 ex

 

The invention relates to the field of biotechnology, medicine and pharmacology, and relates to a method of obtaining a biological drug for medical purposes, namely the isolation and purification of recombinant human growth hormone, secreted by Saccharomyces cerevisiae.

The human growth hormone (somatotropin) is one of the most studied pituitary hormones. Somatotropin is a protein molecule which consists of 191 amino acid residue and contains two intramolecular disulfide bridge. Despite the high degree of homology sequences of growth hormones a variety of mammals, human cells are active only own a hormone or hormone higher primates. Growth hormone is an anabolic hormone, which is essential for postnatal growth, normalization of carbohydrate, lipid, nitrogen and mineral metabolism, tissue regeneration and survival of cells. With the development of recombinant DNA technologies has become possible to create microorganisms - producers of biologically active substances, including human growth hormone. Currently, the use of recombinant human growth hormone is one of the most promising and ever-expanding areas of pharmacology.

Producers of recombinant human growth hormone synthesize it and intracellular and secrete into the environment.

There are many methods of isolation and purification of human growth hormone produced by recombinant strains of microorganisms intracellularly. In particular, the bacteria Escherichia coli form it into cells in the form of Taurus enable (EN 2099348, US 4511503, JP 4112900, CN 101665788), and other organisms accumulate in periplasm or the cytoplasm (US 6436674, EP 0974654, EP 0321940, WO 9419474).

However, for the technology of the target protein using microorganisms-producers, secreting the hormone is preferable because it requires less work.

The allocation method secreted recombinant somatotropin developed for bacteria Bacillus subtilis (Franchi E, Maisano 1991; Teresa M, Ribela 2000), and for the yeast Pichia pastoris, a method of purification of secreted recombinant polyhistidine-containing somatotropin (Calik P, Orman 2008).

There is a method of selection from the culture fluid and purification of recombinant growth hormone secreted by the yeast Pichia pastoris (WO 2010134084), including the two-stage ion-exchange chromatography sorbents type SP-Sepharose FF and Toyopearl SuperQ, hydrophobic chromatography on Toyopearl Butyl Super, filtration on Sephadex G-25 and anionic chromatography on Toyopearl SuperQ. The total yield is 16.8%, and the purity of the obtained protein of 97.8% (according to ivash on C4).

As the closest analogue is selected isolation and purification of recombinant Gorm is on human growth, secreted by Saccharomyces cerevisiae (KR 960013572), including alcohol precipitation of the target protein, dissolving the precipitate in a buffer with urea, ion-exchange chromatography on DEAE-Sepharose at pH 8, the gel-filtration on Sephacryl S-200 or S-300 and re anion-exchange chromatography on DEAE-Sepharose at pH 7.5 (purity of the preparation ≥90%).

The present invention is to expand the Arsenal of methods of isolation and purification of recombinant human growth hormone secreted by the yeast S. cerevisiae.

The task is solved by developing a method for isolation and purification of recombinant human growth hormone secreted by the yeast S.cerevisiae in the process of fermentation in the relevant terms, including the selection of the target protein by the deposition of released from the biomass of the culture fluid by either acidification to pH of 2.9 to 4.0, or adding polyethylene glycol, dissolving the obtained precipitate in a suitable solvent and preliminary purification of the target protein or by anion-exchange chromatography at a pH of 5.6 or method diafiltration in the presence of 0.1-0.5 M sodium chloride, as well as the main cleaning methods anion-exchange chromatography at pH of not less than 7.3 and gel filtration.

The method in General

Use a strain of yeast S.cerevisiae, secreting recombinant human growth hormone in the Fe process is documentation in appropriate conditions. The culture fluid obtained by the fermentation process, are released from biomass and used for selection of the target protein.

The selection of the target protein

Option a

The precipitation of the target protein is carried out by acidification released from the biomass of the culture fluid to a pH of 2.9 to 4.0. The precipitate was separated by centrifugation at 20 thousand rpm for 30 minutes

Option

The precipitation of the target protein is carried out by adding to exempt from biomass cultural liquid solution of polyethylene glycol 3000-6000 Yes to a final concentration of 12-25%. The precipitate was separated by centrifugation at 20 thousand rpm for 30 minutes

Pre-cleaning

Option 1

The precipitate obtained according to option a or option b is dissolved in 50 mm buffer Tris-HCl (pH 7.3)containing 0.025%detergent tween 20 (hereinafter Buffer A), and bring the pH of this solution to a value of 5.6. Then re-centrifuged at 20 Tyson/min for 30 min for further clarification.

Purification of the target protein is performed by the method of anion-exchange chromatography at a pH of 5.6. To do this, the resulting solution was applied to a column of Q-Separate (GE Healthcare Bio-Sciences AB, Sweden), equilibrated with 10 mm Tris-HCl buffer pH of 5.6, containing 0.025%detergent tween-20, and collect the fraction, not adsorbirovavshyei on the column and bring the pH to 7.4 to 8.0. The yield of the target protein, catalyzes and further specify the method iwash on C18 (KR 100274191), up to 65% from the deposition.

Option 2

The precipitate obtained according to option A or option B, is dissolved in a buffer of 10 mm Tris-HCl (pH 7.3)containing 0.025%detergent tween 20 (hereinafter Buffer B).

Purification of the target protein is performed by the method of diafiltration. For this purpose, the solution of the target protein is added an equal volume of 0.4 M sodium chloride in 50 mm Tris-HCl buffer, pH 7.4 and concentrated in 2 times, using a membrane with a cutoff of 10 kDa. Then in the mode diafiltration against 10 mm Tris-HCl buffer, pH 7,4, reduce the concentration of salt in 8 times. The yield of the target protein to 85% from the deposition.

The main purification of the target protein

The main cleaning of the target protein is carried out in two stages.

STAGE 1

For the primary purification of the target protein, subjected to pre-treatment for option 1 or 2, using the method of anion-exchange chromatography at a pH of not less than the 7.3. For this purpose a solution of the target protein is applied to a column of Q-Separate, equilibrated with buffer C. the Column was washed with the starting buffer, then 70 mm solution of sodium chloride in buffer and elute the target protein 0.25 M solution of sodium chloride in the same buffer. The yield of the target protein at this stage is up to 90% for each of the options.

STAGE 2

The final stage of basic cleaning is carried out using gel filtration. For this solution the protein spruce, obtained in STEP 1 is subjected to gel filtration on a column of Superdex G75 (GE Healthcare Bio-Sciences AB, Sweden), equilibrated with buffer containing 0.2 M sodium chloride. The yield of the target protein in a major fraction at this stage is up to 82% for any of the options.

The total yield of the target protein after completion of all stages of cleaning up to 60%.

Example 1. Selection (option A) and treatment (option 1) recombinant human growth hormone, secreted by a strain of the yeast S.cerevisiae VKPM Y-3506

After fermentation, the yeast cells are separated by centrifugation (30 min at 10 rpm). To 100 ml exempt from biomass cultural liquid (supernatant), add 20 ml of 1 M citric acid to achieve a pH of 2.9 and incubated for 1 h at 5°C. the Precipitate was separated by centrifugation (30 min at 20 rpm), dissolved at 5°C in 100 ml of Buffer A and brought to a pH of 5.6 with 0.1 M sodium hydroxide solution. Then re-centrifuged under the same conditions for additional clarification of the solution.

The resulting solution is subjected to pre-treatment. For this purpose it is applied on a column of Q-Separate (10 ml), equilibrated with 10 mm Tris-HCl buffer pH of 5.6, containing 0.025%detergent tween-20. Collect the fraction, not adsorbirovavshyei on the column and bring the pH to 7.4. The yield of the target protein is 58% from the deposition.

Basic clear who the target protein is performed by the method of anion exchange chromatography at pH 7.3 (STAGE 1). For this purpose, the protein solution is applied on a column of Q-Separate, equilibrated with Buffer C. the Column was washed with the starting buffer, then 70 mm solution of sodium chloride in buffer and elute the target protein of 0.25 M solution of sodium chloride in the same buffer. The yield of the target protein at this stage is 85%.

The final stage of basic cleaning is carried out using gel filtration (STAGE 2). To do this, the eluate obtained after anion exchange chromatography, subjected to gel filtration on a column of Superdex G75, balanced with buffer containing 0.2 M sodium chloride. The yield of the target protein at this stage is 82%.

The total yield of the target protein after completion of all stages of purification is 40%.

Example 2. Selection (option A) and treatment (option 1) recombinant human growth hormone, secreted by a strain of the yeast S.cerevisiae VKPM Y-3580

Isolation and purification carried out as in example 1, but as the producer of the target protein using S.cerevisiae strain VKPM Y-3580. The total yield of recombinant human growth hormone after completion of all stages of purification is 38%.

Example 3. Selection (option A) and treatment (option 2) recombinant human growth hormone, secreted by a strain of the yeast S.cerevisiae VKPM Y-3506

The precipitation of the target protein is carried out as in example 1, but the sediment Rast is oraut in 100 ml of buffer A. Pre-cleaning is performed by the method of diafiltration. For this purpose, the solution of the target protein is added an equal volume of 0.4 M sodium chloride in 50 mm Tris-HCl buffer, pH 7.4 and concentrated to 2 times in an Amicon cell (MIllipore, USA) on the membrane DIAFLO PM 10 (Amicon Corporation, USA). Then in the mode diafiltration against 10 mm Tris-HCl buffer, pH 7,4, reduce the concentration of salt in 8 times. The yield of the target protein in the concentrate is 75% from the deposition. Next, conduct a major cleanup of the target protein by the method of anion-exchange chromatography as described in example 1. The yield of the target protein at this stage is 87%. The final stage of basic cleaning is carried out using gel filtration as described in example 1. The yield of the target protein at this stage is 82%.

The total yield of the target protein after completion of all stages of purification is 54%.

Example 4. Selection (option A) and treatment (option 2) recombinant human growth hormone, secreted by a strain of the yeast S.cerevisiae VKPM Y-3580

Isolation and purification carried out as in example 3, but as a producer of the target protein using S.cerevisiae strain VKPM Y-3580. The total yield of recombinant human growth hormone after completion of all stages of purification is 57%.

Example 5. Selection (option B) and treatment (option 1) recombinant human growth hormone, is excretiruemami strain of yeast S.cerevisiae VKPM Y-3506

After fermentation, the yeast cells are separated by centrifugation (30 min at 12 rpm). The precipitation of the target protein is carried out by adding a solution of polyethylene glycol (PEG) 3000 to a final concentration of 25%. For this purpose, 100 ml of supernatant was added 100 ml of a 50%aqueous solution of PEG 3000 and incubated for 30 min at 5°C. the Formed precipitate was separated by centrifugation (30 min at 20 rpm) and dissolved in 100 ml of buffer A. Further purification carried out as in example 1.

The total yield of the target protein after completion of all stages of purification is 44%.

Example 6. Selection (option B) and treatment (option 1) recombinant human growth hormone, secreted by a strain of the yeast S.cerevisiae VKPM Y-3580

Isolation and purification carried out as in example 5, but as the producer of the target protein using S.cerevisiae strain VKPM Y-3580. The total yield of recombinant human growth hormone after completion of all stages of purification is 42%.

Example 7. Selection (option B) and cleaning (option 2) recombinant human growth hormone, secreted by a strain of the yeast S.cerevisiae VKPM Y-3506

After fermentation, the yeast cells are separated by centrifugation (30 min at 12 rpm). The precipitation of the target protein is carried out by adding a solution of PEG 6000 to a final concentration of 17%. For this to 100 ml super is atanta add 50 ml of 50%solution of PEG 6000 and incubated for 30 min at 5°C. The formed precipitate was separated by centrifugation (30 min at 20 rpm) and dissolved in 100 ml of buffer A.

Cleaning is carried out as in example 3. The total yield of recombinant human growth hormone after completion of all stages of treatment is 60%.

Example 8. Selection (option B) and cleaning (option 2) recombinant human growth hormone, secreted by a strain of the yeast S.cerevisiae VKPM Y-3580

Isolation and purification carried out as in example 7, but as the producer of the target protein using a strain of S. cerevisiae VKPM Y-3580. The total yield of recombinant human growth hormone after completion of all stages of purification is 58%.

Thus, the inventive method allows to obtain recombinant human growth hormone with the yield up to 60%that is significantly higher yield (17%), known for yeast producer Pichia pastoris.

With the purity of the drug target protein, obtained by the claimed method (up to 100% according to the method iwash on C18 column) corresponds to the purity (up to 100%, according to the method iwash in column C4) drug obtained using yeast producer Pichia pastoris (WO 2010134084) and exceeds the purity of the product obtained by the method closest analogue (KR 960013572).

Sources of information

1. EN 2099348.

2. US 4511503.

3. JP 4112900.

4. CN 101665788.

5. US 6436674.

6. EP 0974654.

7. EP 0321940.

8. WO 9419474.

9. A new human growth hrmone production process using a recombinant Bacillus subtilis strain. Elisabetta Franchi, Federico Maisano, Silvia Astrua Testori, Giuliano Galli, Salvatore Toma, Luca Parente, Francesca de Ferra, Guido Grandi, J. Biotechnology, V.18, Issues 1-2, April 1991, pages 41-54.

10. Synthesis and chromatographic purification of recombinant human pituitary hormones. Maria Teresa C.P. Ribela, Peter W. Gout, Paolo Bartolini; J. Chromatography B, V.790, Issues 1-2, pages 285-316.

11. Expression system for synthesis and purification of recombinant human growth hormone in Pichia pastoris and structural analysis by MALDI-ToF Mass Spectrometry. Calik P., M.A. Orman, Celik E., Halloran S.M., Calik G., Ozdamar T.N., Biotechnol. Prog.; 2008 Jan-Feb; 24(1):221-6. Epub 2008 Jan 11.

12. WO 2010134084.

13. KR 960013572.

14. KR 100274191.

The isolation and purification of recombinant human growth hormone, secreted by Saccharomyces cerevisiae during fermentation under appropriate conditions, including the selection of the target protein from liberated from the biomass of the culture liquid by sedimentation, dissolving the obtained precipitate in a suitable solvent and primary purification of the target protein by the methods of anion-exchange chromatography by sorption him at a pH of not less than 7.3 is followed by elution and gel filtration, characterized in that the deposition is carried out either by acidification to pH of 2.9 to 4.0, or the addition of polyethylene glycol 3000-6000 Yes, and besieged the target protein before the main cleaning is subjected to pre-treatment or by passing in pH of 5.6 through a column of anion-exchange sorbent, or method of diafiltration against 0.1-0.5 M sodium chloride.



 

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