RU2048521C1 - Method of preparing polypeptide showing human lymphotoxin property - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: strain of E. coli ВКНМ B-5279 showing high level of polypeptide synthesis and property of human lymphotoxin is used as a source of the end product. After cultivation of strain-producer, cell rupture and separation of cellular residue two-stage chromatographic purification of extract is carried out (on DEAE-cellulose at pH 8.7-9.2 and hydroxyapatite at pH 6.6-6.8). EFFECT: high yield of homogenous human lymphotoxin preparation. 5 tbl

Description

 The invention relates to biochemistry and biotechnology, and is a method of isolating and purifying a physiologically active substance from a producer strain containing recombinant plasmid DNA encoding a polypeptide with the properties of human lymphotoxin, and can be used to produce a polypeptide for the purpose of a detailed study of its properties and use in medical practice.

 In the human body, lymphotoxin (RT, aka tumor necrosis factor-beta, TNF-β) is produced mainly by activated T-lymphocytes. It is a glycoprotein with a mol.m. 25,000 Yes, it was first discovered in the late 60s. In vitro and in vitro experiments have shown that natural (glycosylated) and recombinant (non-glycosylated) and recombinant (non-glycosylated) lymphotoxin causes hemorrhagic necrosis of some types of solid tumors. Like tumor necrosis factor, α-lymphotoxin, being a broad-spectrum immunomodulator, exhibits its cytotoxic activity highly selectively, affecting only tumor cells and not affecting healthy, untransformed cells. In addition, lymphotoxin has a pronounced antiviral effect. All this makes the use of lymphotoxin in medicine extremely promising.

 For a deep study of the properties of lymphotoxin, its biological and medical tests, a significant amount of the drug is required, and therefore, it is necessary to create a simple and effective method for its isolation and purification.

A known method of producing LT from an activated lymphoblastoid human cell line RPMi-1788. The method includes concentrating the intercellular fluid, chromatography on DEAE cellulose, preparative isoelectric focusing, chromatography on lectin-sepharose and preparative polyacrylamide gel electrophoresis. Yield: 25 μg of electrophoretically homogeneous RT from 100 L of cell culture, which is about 11% of the initial content of LT in cell culture. The specific cytolytic activity of the obtained preparation was 4 × 10 ⁷ units / mg.

 The disadvantages of the method: the following complex, laborious methods of culturing the cell line and purification of the drug, low yield of the target product.

 Known methods for producing lymphotoxin microbiological synthesis using recombinant plasmids. The method is based on the purification of recombinant RT using column chromatography with monoclonal antibodies. There is no data on the yield of the target product.

 The disadvantages of the method: the following complexity and high cost of obtaining an immune sorbent, the difficulty of scaling the process, the need for additional control of the target product for the absence of oncogenic material of the parent myeloma cell used to obtain monoclonal antibodies.

The method is based on the use of E. coli strain HB 101 transformed with plasmid pCG 402 containing the TNF-β gene under the control of the tryptophan promoter. Cells are grown on glucose-salt medium with the addition of casamic acids, thiamine, tryptophan and ampicillin. Indole acrylic acid is induced to express the TNF-β gene. The obtained cells are destroyed by ultrasound and the cell extract is chromatographed on DEAE-Sepharose CL-6B. TNF-β is eluted in a Tris-HCl balancing buffer, pH 7.5, containing 30 mM NaCl. To remove impurities of the bacterial endoxin, a detoxigel column is used. The yield of recombinant TNF-β was 8 mg from 1 L of cell culture; the specific activity of the drug was 3-5 ˙10 ⁷ u / mg.

The disadvantages of the method are as follows: the use of deficient additives thiamine, tryptophan at the producer’s growing stage; the use of an inducer for expression of the target gene; A fast, almost one-step process of purification of TNF-β is possible only with a high content in the total mass of intracellular proteins. The method used cells with a target product content of up to 34% of the total mass of cellular proteins, however, 10-12% TNF-β was in soluble form
The closest (prototype) to the claimed method is a method for purification of a recombinant human tumor necrosis factor. The method is based on the use of E. coli strain M15 transformed with plasmid pDS 78 / RBS11 encoding TNF-β. The cells are grown in a standard way. To express the TNF-β gene, the lactose operon isopropyl-1-thio-β-D-galactopyranoside is induced. The obtained cells (60 g with 10 L of medium) are suspended in 50 mM Tris-HCl pH 8.5 buffer containing 10% glycerol, 5 mM dithiothreitol, 10 mM benzamidine chloride, 1 mM orthophenanthroline, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 2 , 8 mg of DNase and 100 u / ml of trasilol, are destroyed by ultrasound. Insoluble material was removed by centrifugation, the supernatant was dialyzed against 50 mM Tris-HCl pH 8.5 buffer containing 1 mM EDTA and passed through a DEAE-Sepharose column. Elution is carried out with the same buffer. Fractions were analyzed for LT content by gel electrophoresis in a 15% polyacrylamide gel in the presence of Lemmley SDS. RT fractions are collected and applied to an S-Sepharose column. Elution is carried out with a linear NaCl gradient from 0 to 1 M in 50 mM Tris-HCl buffer pH 8.5. LT elutes at a NaCl concentration of about 200 mM. Then LT is purified by fractionation with ammonium sulfate, precipitating the target product at 20% salt saturation. The precipitate was separated by centrifugation and dissolved in buffer. The yield of electrophoretically homogeneous RT was 10 mg from 10 L of cell culture (0.17 mg from 1 g of biomass). The specific cytolytic activity of the drug is comparable to the cytolytic activity of the standard preparation of TNF-α (absolute value not shown).

The disadvantages of the prototype method are as follows: the use of strain E. coli M-15, requiring the induction of the lactose operon isopropyl-1-thio-β-D-galactopyranoside and containing proteases of various types, capable of inactivating RT during the cleaning process, and, as a consequence, the use for protection against proteolysis of a set of protease inhibitors benzamidine chloride, orthophenanthroline, phenylmethylsulfonyl fluoride and trasilol;
the use of insufficiently effective cleaning methods elution of LT by long-term washing with a buffer with low ionic strength, at pH 8.5, practically without sorption on DEAE-sepharose, which leads to the difficulty of separating the target product from ballast proteins;
the use of S-Sepharose, which does not allow to obtain a sufficiently homogeneous product;
the use of fractionation with ammonium sulfate, leading to loss and reduction of product yield.

 The aim of the invention is to simplify the method of purification of lymphotoxin and increase the yield of the target product.

 This is achieved by using in the method E. coli strain VKPM B-5279, transformed with a plasmid encoding a polypeptide with human lymphotoxin properties, together with purification of lymphotoxin by ultrasound disintegration of cells, separation of cell debris by centrifugation and subsequent cell extract chromatography on DE-52 DEAE cellulose at pH values of 8.7-9.2 and hydroxylapatite at pH values of 6.6-6.8.

 The essence of the method is as follows.

The biomass of E. coli cells VKPM B-5279, grown in a standard way in L-broth and collected by centrifugation, is suspended in buffer A (10 mm Tris-Hcl 1 mm EDTA, pH 8.7-9.2) in a ratio of 4-5 ml buffer per 1 g of cells, add phenylmethylsulfonyl fluoride to 0.1 mm. The suspension is subjected to sonication to reduce the optical density at a wavelength of 550 nm to 50-55% of the original. Cell debris is removed by centrifugation and the supernatant is applied onto a DE-52 DEAE cellulose column (based on 1 g of cells, 4-4.5 ml of sorbent). The column is washed with buffer A until the optical density in the effluent is reduced at a wavelength of 280 nm to the initial value and the proteins are eluted with a linear gradient of NaCl concentration in buffer A. The lymphotoxin solution eluted from the column at a concentration of 0.07-0.12 M NaCl is diluted in 1, 5 times with buffer B (10 mM HEPES, pH 6.6-6.8) three times the concentration, the solution is triturated to a final pH of 6.6 and applied to a hydroxylapatite column. The column was washed with buffer B and the proteins were eluted with a linear NaCl gradient. Lymphotoxin preparation eluted from the column at 0.15-0.3 M NaCl concentration has a purity of about 95%
The yield of electrophoretically homogeneous LT is 25-35% of the content in the original cell extract. When the content of LT in the producer in the amount of 10% of the total amount of cellular proteins is 1.8 mg of 1 g of biomass (10 m / l cell culture), which is 10 times higher than the yield of the product according to the prototype method. The specific activity of the resulting preparation is not less than 3 107 units / mg of protein (according to the cytolytic effect on the cells of murine fibroblasts L 929 in the presence of actinomycin D).

 A new feature compared to the prototype method is the use of E. coli strain VKPMV-5279 in conjunction with chromatographic purification of the target product on DEAE cellulose at pH 8.7-9.2 and on hydroxylapatite at pH 6.6-6.8 . It is this combination of distinguishing features that provides a highly purified preparation of lymphotoxin, which is necessary for the needs of medicine.

 The use of E. coli strain VKPM B-5279 in the method with fewer proteases than in the prototype allows not to use deficient reagents such as protease inhibitors benzamidine chloride, orthophenanthroline, trasilol. The use of a plasmid carrying the polypeptide gene with the properties of human lymphotoxin, which provides constitutive synthesis of RT, does not require induction for expression. Chromatography of the cell extract is carried out on DEAE cellulose in a buffer with a concentration 5 times lower than in the prototype and pH values of 8.7-9.2, which leads to sorption of the target product on a sorbent, and elution is carried out with a sodium chloride gradient. This allows you to most effectively clean the target product from ballast proteins without significant losses. At pH values below 8.7 and above 9.2, a significant portion of LT begins to elute in the application buffer along with the bulk of the ballast proteins, which leads to a decrease in the cleaning efficiency and loss of the target product.

 The second chromatographic purification of RT on hydroxylapatite in a buffer with pH values of 6.6–6.8 also allows the main part of the product to be sorbed on a column, freed from non-absorbable ballast proteins and eluted highly purified RT in a salt concentration of 0.15–0.3 M with a high output (table 1.2).

 The proposed cleaning method allows to obtain an electrophoretically homogeneous product with a yield of 25% from cells in which the LT content is only 0.5% (Table 3), which indicates its effectiveness. The method is easily scalable.

PRI me R 1. Obtaining a highly purified preparation of lymphotoxin 10 cells (E. coli VKPM B-5279) are suspended in 40 ml of buffer A (10 mm Tris-Hcl, 1 mm EDTA, pH 8.8) with the addition of phenylmethyl sulfonyl fluoride to 0 , 1 mm to a homogeneous state. Then the suspension is subjected to ultrasonic treatment (UZDN-2T; A-24; t≅ 5 ^о С) 3-4 times for 25-30 s until the optical density decreases at a wavelength of λ ₅₅₀ nm to 50-55% of the initial one. The cell debris was removed by centrifugation at 13500 rev / min for 90 min (JA-21; JA-14 rotor; t 2 ° ^C) and the supernatant was applied to a column (1.6 20 cm; V 40 mL) with DEAE-cellulose de- 52 equilibrated with buffer A. The column is washed with buffer A until the optical density in the effluent decreases at a wavelength of λ ₂₈₀ nm from the initial value and the proteins are eluted with a linear gradient of NaCl concentration from 0 to 0.5 M in buffer A (V _gradient 200 ml). The analysis of fractions for the content of LT in them is carried out by gel electrophoresis in a 15% polyacrylamide gel in the presence of 0.1% SDS, according to Lemmli.

Lymphotoxin is usually eluted from the column at 0.07-0.12 M NaCl concentrations. RT fractions are combined (V40 ml), 1/2 volume (20 ml) 30 mm HEPES buffer, pH 6.6, under pH control is added the meter, the pH is adjusted to 6.6 using a 0.1 M HCl solution. After this, the solution is applied to a column (2.6 × 5 cm; V 26 ml) with hydroxylapatite, equilibrated with buffer B (10 mM HEPES, pH 6.6). The column is washed with buffer B until the optical density in the effluent is reduced at a wavelength of λ ₂₈₀ nm to the initial one and the proteins are eluted with a linear gradient of NaCl concentration from 0 to 0.5 M in buffer B (V _gradient, 150 ml).

Lymphotoxin is usually eluted at a NaCl concentration of 0.15-0.3 M. Fractions containing lymphotoxin are pooled and dialyzed against 0.010 M Na ₂ HPO ₄ buffer, pH 7.2, 0.15 M NaCl, final volume 26 ml.

The yield of lymphotoxin is 18.3 mg. The protein concentration is 0.7 mg / ml. The activity of the drug is 5 107 units / mg. Lymphotoxin purity> 96%
PRI me R 2. Chromatographic purification of lymphotoxin from cell extract DEAE-cellulose DE-52 at various pH values.

 Purification of the drug lymphotoxin is carried out as in example 1, except that the elution of the proteins is carried out at pH values from 7.6 to 9.6. The cleaning results are presented in table 4.

 From the data in Table 4, it is seen that during chromatographic purification of LT on DEAE cellulose at a pH above 9.2 and below 8.8, the proportion of the product eluted when applied to the column increases, while its amount in the target fraction eluted with sodium chloride decreases and a purification factor during chromatography from 2 to 1.8 at pH 9.6 and 1.6 at pH 8.4.

 PRI me R 3. Chromatographic purification of lymphotoxin on hydroxylapatite at various pH values.

 Purification of the drug lymphotoxin on hydroxylapatite is carried out as in example 1, except that the elution of the product is carried out with an appropriate buffer at pH values from 6.3 to 7.4. The cleaning results are presented in table.5. In experiment 8, the elution is carried out with a gradient of potassium phosphate.

 The table shows that when chromatographic purification of RT on hydroxylapatite at a pH above 6.8 and below 6.6 increases the proportion of the product that is not adsorbed onto the column and eluted with ballast proteins. This reduces the amount of product in the target fraction eluted with sodium chloride, and reduces the cleaning factor.

Thus, the use of the proposed method in comparison with the prototype allows to exclude from the process deficient reagents proteazbenzamidine chloride, orthophenanthroline, trisilol inhibitors; isopropyl-1-thio-β-D-galactopyranoside inducer of the lactose operon;
to simplify the process of culturing cells of the producer strain by eliminating the stage of induction of the synthesis of the target product;
to increase the yield of the target product 10 times (RT yield from 1 g of biomass 1.8o mg, in the prototype 0.17 mg from 1 g of biomass);
apply the purification method for the production of RT from biomass with the content of the target product from 0.5 to 10% of cellular proteins.

 These advantages were achieved by using the E. coli VKPM B-5279 producer strain in the method together with purification of lymphotoxin by chromatography on DE-52 cellulose DE-52 at pH values of 8.7-9.2 and chromatography on hydroxylapatite at pH 6.6- 6.8.

Claims (1)

 METHOD FOR PRODUCING A POLYEPEPTIDE WITH PROPERTIES OF HUMAN LYMPHOTOXIN, including culturing a producer strain, destroying cells with ultrasound, removing cell residue and chromatography on sorbent columns, characterized in that Escherichia coli VKPM B-52 is used as a source of lymphotoxin to purify chromatograph A 79 chromatography. sequentially on DEAE cellulose at pH values from 8.7 to 9.2 and on hydroxylapatite at pH values from 6.6 to 6.8.

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