RU2055895C1 - METHOD OF PREPARING RECOMBINANT HUMAN α1 THYMOSINE - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves the transformation of the strain E. coli SG20050 with plasmid pThy 315 encoding hybrid protein α₁-factor of tumor necrosis - α thymosine. Transformants were cultured on the liquid nutrient medium and isolation and splitting hybrid protein is carried out with cyanogen bromide in the medium of formic and trifluoroacetic acids. Prepared inclusive bodies were washed with 1-3 M urea solutions and thymosine is separated from protein components by pH medium change to 5-6 and removal of precipitate formed. Thymosine is purified successively by sieve, ion-exchange and phase-reversed chromatography methods. Carrier of type "Sephadex G-25" is used for sieve chromatography; carrier of type DE-, DEA- or DEAE is used for ion-exchange chromatography (at pH 6.8 in sodium-phosphate buffer containing 10% acetonitrile); carrier of type 08-C18 is used for phase-reversed chromatography where product is eluted at acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. Method provides the preparing hybrid protein with 99% purity, not less. EFFECT: high purity of product.

Description

The invention relates to biotechnology, in particular, to a method for microbiological synthesis in recombinant peptide with Escherichia coli cells with the properties of human thymosin α ₁ .

There is a method of producing thymosin α ₁ by large-scale extraction of peptides from calf thymus followed by chromatographic isolation of the target peptide [1] The disadvantage of this method is the need to slaughter a large number of newborn calves, since the thymosin α ₁ in thymus has the highest content in calves up to 6 months and is about 10 mg / kg of gland. With increasing age of animals, the content of thymosin a decreases sharply, reaching less than 1 mg / kg. In addition, to obtain a sufficiently pure peptide, repeated use of chromatographic methods is necessary, which greatly complicates the technology.

A known method for producing recombinant thymosin α ₁ from a hybrid protein with β -galactosidase encoded by the plasmid pThα ₁ β gal with the LacUY5 promoter in E. coli strain 294. The hybrid protein was cleaved with cyanogen bromide in 88% formic acid, and thymosin α _{1 was} isolated by sequential chromatography on DE-52, gel filtration, and a C-18 sorbent. The yield of thymosin α ₁ was 1 mg / l of culture medium or 400 μg / g of cells [2]
Known closest to the proposed method for the preparation of thymosin α ₁ using the recombinant plasmid pThy314 encoding the synthesis of a hybrid protein TNF-thymosin α ₁ , from which thymosin can be cleaved with bromine hydrolysis, and E. coli strain SG20050 (pTh314), which provides the expression level of hybrid protein equivalent to 8-10 mcg of thymosin in one ml of cell susepenzii at a density of 10 ⁹ cells / ml of [3] The disadvantages of the process described in the prior art, are: the presence in corpuscles tsitoplazmatichekih cells along with the inclusion of the fusion protein of TNF-t mozin in an equal amount by weight of chloramphenicol acetyl transferase protein, which greatly complicates the process of purification of the fusion protein; In addition, the plasmid pThy 314 is unstable during storage of strain SG20050 (pThy314) on solid nutrient media, which leads to a low level of synthesis of the hybrid protein; the hybrid protein in 80% formic acid and 0.1% bromine is cleaved by 50-60%, which reduces the yield of thymosin α ₁
The objective of the invention is to provide a more productive and simple method for producing recombinant thymosin α ₁ .

The problem is solved in that a method for producing recombinant human thymosin α _{1 is} proposed, comprising transforming the Escherichia coli strain SG20050 with a plasmid encoding a hybrid protein α-tumor necrosis factor-thymosin-α ₁ culturing the transformant in a liquid nutrient medium, isolating and cleaving the hybrid protein with bromine cyan formic acid medium, the separation of thymosin α ₁ from the protein components of hydrolysis and its chromatographic purification; moreover, plasmid pThy315 obtained after isolation of the body was used for transformation and the inclusions are washed with 1-3 M urea solutions and water, and the fusion protein is cleaved in the presence of 0.1 M trifluoroacetic acid, thymosin is separated from the protein components of the hydrolyzate by changing the pH of the medium to 5-6 and removing the precipitate that forms, chromatographic purification of the target product is carried out sequentially on Sephadex G-25, on a DEA-type ion-exchange sorbent at pH 6.8, in sodium phosphate buffer containing 10% acetonitrile, and reverse phase chromatography on a C ₈ -C ₁₈ sorbent with thymosin elution α ₁ gra a acetonitrile diet in the range of 16-25% in the presence of 0.1% trifluoroacetic acid.

The following technical results are achieved:
the only major hybrid protein is synthesized in E. coli cells, which facilitates the cleaning process;
fusion protein reproducibly produced as an insoluble precipitate of about 70% purity;
the use of a mixture of formic and trifluoroacetic acids allows you to dissolve the hybrid protein and increase the efficiency of its bromocyanine hydrolysis to 90%, which is not achieved by using these acids separately;
chromatography of a hydrolyzate on a Sephadex G-25 gel results in an effective separation of a significant proportion of impurities from thymosin, which, in turn, allows the thymosin fractions to be concentrated by simple evaporation of the solution in vacuo, to reduce the volume of chromatographic columns in subsequent stages, and therefore to speed up and reduce the cost of separation on them;
the chromatographic system used in the last stage contains volatile components that are removed by simple evaporation under vacuum and do not require additional stages of converting the product into the free acid form.

The proposed cleaning method allows to obtain deacetylmosin α ₁ not less than 99% purity according to electrophoresis and HPLC. The output of thymosin per 1 g of fusion protein is not inferior to that described in the prototype. The total time saving for one selection cycle is at least 12-14 hours;
in General, the proposed method is more productive, fast and technologically advanced.

PRI me R. Obtaining thymosin α _{1 by} microbiological synthesis in E. coli cells.

The plasmid DNA pTU315 was transformed into competent cells of E. coli strain SG20050. Transformed cells were inoculated into broth containing 50 .mu.g / ml ampicillin and cultured at ³⁷ ° C for 18-20 hours. The grown cells were assayed for the presence of fusion protein in 15% SDS-PAGE. Cells of strain SG20050 with plasmid pThy315 are pelleted by centrifugation at 3000 g for 10 minutes. The broth is drained, the cells are suspended in a lysis buffer containing 20 mm Tris-Hcl, pH 7.5 mm Na ₂ EDT and 1 mm phenylmethylsulfonyl fluoride, based on 1 g of cells 10 ml of buffer. Cells are destroyed in any way (for example, by freezing-thawing or ultrasound, pulses of 40 s three times, or by forcing in a frozen state through a die in a French press or X-press). The lysate was centrifuged for 10 min at ⁴ ° C, 5000g. The supernatant is drained, a solution of 1 M urea is added to the residue at the rate of 10 ml per 1 g of cells, and intensively mixed. Repeat centrifugation. The supernatant is drained, the precipitate is suspended in a solution of 2 M urea of the same volume. Repeat centrifugation. The supernatant is drained. The wet protein precipitate is washed in the same way with water.

The precipitate is dissolved in 70% formic acid, 10% by volume of 1.1 M trifluoroacetic acid and bromine cyan are added at the rate of 20 ml of formic acid solution and 0.1 g of bromine cyan per 1 g of wet cake. Cleavage is carried out at room temperature for a day. Thereafter, the hydrolysis mixture was diluted with five volumes of water and concentrated on a rotary evaporator at 40 ^{° C,} repeating the dilution and evaporation 2-3. The solution is neutralized with 1M sodium hydroxide or 30% ammonia solution until a pH of 5-6 is reached. The precipitate was separated by centrifugation and the supernatant was concentrated on a rotary evaporator at 40 ^{° C} and applied to a column of 150 ml Sephadex G-25. After elution with water at a rate of 2 ml / min substance first peak was collected and evaporated on a rotor evaporator under vacuum at 40 ^C to a volume of 1-2 ml and applied to a high performance anion exchange column "Silasorb DEA" 8 h 250 mm (manufactured SP "BIOHIMMAK" , Moscow), balanced with 100 mm sodium phosphate buffer, pH 6.8, containing 10% acetonitrile. The column is eluted with this buffer at a rate of 2 ml / min with detection at 225 nm. Thymosin the chromatogram corresponds to the main peak, which is collected, concentrated by evaporation under vacuum ^at 40 ° C until the onset of crystallization content and desalted on a column of Sephadex G-25. Thymosin fractions are collected, concentrated by evaporation, as described above, to a volume of 0.5-1 ml and chromatographed on a high-performance column "Diasorb S-16" 4x25 mm (produced by Biokhimmak SP, Moscow) in a reverse phase gradient system 16-25% in the presence of 0.1% trifluoroacetic acid with an elution rate of 1 ml / min. Fractions of the main peak constituting bolmee 80% of the total absorbance applied substance was concentrated by evaporation under vacuum ^at 40 ° C and lyophilized. A homogeneous preparation is obtained chromatographically and electrophoretically (when analyzed in SDS-PA-AG). The presence of a serine precipitate at the N-terminus of the oligopeptide chain, as well as the correspondence of the amino acid composition to the expected one, is confirmed by amino acid analysis of the isolated recombinant thymosin. The results of determining the amino acid composition of the product after hydrolysis of 6 N HCl with the addition of 0.2% phenol at 110 ° C for 24 hours on an Alpha Plus analyzer (LKB) are as follows: Asp 4.64 (4); Thr 2.86 (3); Ser 2.86 (3); Glu 6.78 (6); Ala 3.0 (3); Val 3.03 (3); Cys 4.30 (4); Leu 2.3 (2).

Claims (1)

METHOD FOR PRODUCING HUMAN RECOMBINANT THYMOSINE α ₁ , including transformation of Escherichia coli strain SG 20050 with a plasmid encoding the fusion protein α-tumor necrosis factor thymosin α ₁ , culturing the transformant in a liquid nutrient medium, isolating and cleaving the hybrid protein with bromine acid α ₁ from the protein hydrolyzate components and the chromatographic purification, characterized in that the plasmid used for transformation pThy 315 obtained after separation of the inclusion bodies washed with 1 - 3 M pa urea and water, and the cleavage of the hybrid protein is carried out in the presence of 0.1 M trifluoroacetic acid, thymosin is separated from the protein components of the hydrolyzate by changing the pH of the medium to 5-6 and removing the formed precipitate, chromatographic purification of the target product is carried out sequentially on Sephadex G-25, on a DEA-type ion-exchange sorbent at pH 6.8 in sodium phosphate buffer containing 10% acetonitrile and reverse phase chromatography on a С ₈ - С _{1 8} sorbent with elution of thymosin α ₁ with an acetonitrile gradient in the pre elax 16 - 25% in the presence of 0.1% trifluoroacetic acid.