Method for preparing highly purified recombinant polypeptide with property of human tumor necrosis factor-alpha

FIELD: biotechnology, preparative biochemistry.

SUBSTANCE: invention proposes a method for preparing the recombinant human tumor necrosis factor-aplha (TNF-alpha). Method involves culturing the strain-producer E. coli SG20050/pTNF311Δ, disruption of cells by ultrasonic oscillation, extraction of the end protein and 3-step chromatography purification procedure on DEAE-cellulose column in the linear gradient concentrations of NaCl at pH 8.0, on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 8.0 and on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 6.7. The recombinant TNF-alpha prepared by the proposed method shows the reduced content of impurity proteins, nucleic acids and lipopolysaccharides especially that provides its direct medicinal using. Invention can be used in medico-biological industry.

EFFECT: improved preparing method, enhanced quality of polypeptide.

2 tbl, 5 dwg, 2 ex

 

The invention relates to biochemistry and biotechnology and is a way of allocating and ultracide physiologically active recombinant polypeptide with properties of tumor necrosis factor alpha (TNF-alpha) the person received an engineered way to develop on the basis of medical drug.

TNF-alpha is produced in the body activated macrophages and is a polypeptide consisting of 157 amino acid residues, with a molecular weight of about 17,000 Yes. During the study of its properties in vitro, it was shown that it is able to cause lysis of tumor cells of various lines and no effect on the normal cells of the body and also plays a key role in immune and inflammatory reactions [1-3].

Deep and wide-ranging study of TNF-alpha, and other physiologically active eukaryotic proteins became possible thanks to the technique of recombinant DNA, which produce the polypeptide in large quantities by microbiological synthesis in E. coli cells, transformed with the plasmid carrying the gene for TNF-alpha man. At the present time all over the world are conducted clinical trials on the basis of recombinant TNF-alpha as an antitumor means and means for the treatment of severe viral infecti the (psoriasis, hepatitis b) [4-6].

One of the main problems when developing a method of producing recombinant TNF-alpha is an increase in the yield of the target protein from the source cell extract. This task biotechnology is successfully solved. A method of obtaining recombinant TNF-alpha, providing 63%yield from its content in the original extract of cells of the producer strain [7].

When you create drugs based on recombinant TNF-alpha is critical is the purity obtained by microbiological synthesis of the substance. The content of DNA, lipopolysaccharide (LPS) and bacterial impurity proteins in recombinant proteins used in medicine, is highly undesirable and is strictly regulated: the DNA content should be no more than 100 PG/mg protein, and the content of LPS and proteins producer strain is not more than 200 ng/mg protein [8, 9]. Than below the specified parameters, the drug is less toxic, and, therefore, more efficient.

Known methods for the efficient purification of recombinant TNF-alpha, using affinity chromatography on a column with monoclonal antibodies [10, 11]. Data output product no. The disadvantages of these methods are the following: the complexity and high cost of obtaining immune sorbent, the difficulty scaling process, the need for more to the control of the target product in the absence of a carcinogenic material parental myeloma cells, used to obtain monoclonal antibodies.

There is a method of obtaining and purification of the recombinant polypeptide with properties of TNF-alpha man, based on the use of strain E. coli SG20050 transformed by the plasmid pTNF31Δcarrying the gene for TNF-alpha under the control of the promoters of the early region of bacteriophage T7, providing constitutive synthesis of the target protein [12]. According to this method the destruction of cells of strain-producer conduct ultrasound in hard mode - 4 cycles scoring by 5 min, allowing the heating of the treated biomass to 50°C. Such treatment promotes deep destruction of cells and cellular structures and in the solution of all the components of cells, including nucleic acids (DNA, RNA), (LPS) and associated cellular proteins, which is necessary to purify the target protein as closely as possible. A large number of these impurities in the substance of TNF-alpha produced by this technology, which makes the purification of the recombinant polypeptide intractable. In the method using four different sorbent: neutral N-γgel, aminoalkenes - QAE-γgel, cation-exchanger - R-γgel and Sephadex G-25, which makes the cleaning process is multi-step and time consuming. As a buffer system by chromatography on P-γ-gel use expensive is Activ - citric acid (OSC). In addition, for clarification of cell extract according to the proposed method requires special expensive equipment import ultrafiltration unit. The release of TNF-alpha is low and is only 47% of the content in cell extracts. The degree of purity of the preparation was assessed only according to the electrophoresis, the content of impurities FSC cellular nucleic acids and proteins, as well as progenote drug is not defined.

The closest was effective and technical essence is a way [13; prototype]. According to the method prototype biomass of cells of E. coli SG20050/pTNF311Δgrown in a standard way in L-broth and collected by centrifugation, suspended and subjected to sonication in a milder conditions (5-6 times at 1 min intervals between treatments of 1 min). Cellular debris is removed by centrifugation and carry out the chromatographic purification of the target product on two commercially available sorbents: DEAE-cellulose and hydroxyapatite. To this supernatant is applied on the column with DEAE-cellulose DE-52 and washed with buffer A (20mm Tris-HCl, 0.5 mm EDTA pH 7.5 to 7.7), proteins elute with a linear concentration gradient of NaCl from 0 to 0.15 M in buffer A. Suirvey column with a solution of TNF-alpha at a concentration of NaCl Of 0.07-0.08 M diluted with two volumes of the buffer a and applied to a column with hydroxyapatite, which was washed with buffer (0.05 M K-phosphate pH of 6.8 to 7.0), proteins elute with a linear concentration gradient of K-phosphate from 0.05 to 0.33 M Solution of TNF-alpha, suirvey with a column when the concentration of K-phosphate of 0.28-0.30 M, has a purity of about 87%. Product cialiswhat against buffer a and subjected to re-chromatography on a column with DEAE-cellulose DE-52, as described above.

The drug TNF-alpha, suirvey with sorbent at a concentration of NaCl 0,07 -0,08 M, has a purity of about 95%. Output electrophoretic homogeneous preparation TNF-alpha is 50-60% of the content in the source cell extracts. When the content of the target protein producer in the amount of 24% of the amount of cellular proteins specific productivity is 8.6 mg from 1 g of cells. The content of impurity proteins producer strain in the final product, as determined by ELISA [9], is approximately, but not more than 200 ng/mg TNF-alpha; impurity content of DNA determined by molecular hybridization [15], is not more than 100 PG/mg TNF-alpha; the content of LPS, a certain chemical-enzymatic method [14], about 200 ng/mg TNF-alpha.

Technology for the prototype method is effective, provides a relatively high yield of the target protein (50-60%). However, the purity of the product, characterized by indicators of the quality requirements of medical preparations, obtained by mikrobiologisches the synthesis (the content of impurity proteins of cells of strain-producer, nucleic acids, LPS), is permissible.

An object of the invention is to develop a method of producing ultrapure preparation of physiologically active recombinant polypeptide with properties of TNF-alpha with reduced content of impurity components suitable for use in medicine, while maintaining a consistently high yield of the target product from biomass producer strain.

The problem is solved by using the method of E. coli strain SG20050 transformed by the plasmid pTNF311Δthat encodes a polypeptide TNF-alpha man 5th to 157 amino acid under the control of two promoters of the early region of the T7 phage [12] in conjunction with the purification of the target polypeptide by disintegration of cells by ultrasound, Department of cell debris by centrifugation, additional extraction of the target protein by washing the cell debris buffers, three successive stages of chromatographic purification of extracted proteins on diethylaminoethylcellulose at pH 8.0 and hydroxyapatite at pH 8.0 and then at pH of 6.75. Increasing the pH to 8.0 with the application of a cell extract on diethylaminoethylcellulose provides a more durable binding of TNF-alpha with the carrier and excludes partial loss during application and rinsing of the column.

The method of the bookmark is reported in the following.

The biomass of E. coli cells SG20050/pTNF311Δgrown in katalozhnyh flasks or in a fermenter in L-broth and collected by centrifugation, suspended in the buffer to the fracture (25 mm Tris-HCl, pH 8.0±0,1, 1 mm EDTA, 1 mm PMSP) in the ratio of 7 ml of buffer per 1 g of biomass. Then the suspension was placed in an ice bath and subjected to ultrasonic treatment (installation of UZDN-2T) at a frequency of 22 kHz 5 times at 1 min intervals for 1 min for cooling of the mixture (the temperature should not exceed 8±2°). Cellular debris collected by centrifugation for 50 min at 16000 rpm (centrifuge J2-21, rotor JA-20, the temperature of 4±2°C), washed with buffer to destruction, then separate the phases by centrifugation as described above. United supernatant applied to a column of DEAE-cellulose DE-52 (8-11 mg protein per 1 ml sorbent). The column was washed with buffer A (25 mm Tris-HCl, 1 mm EDTA, pH 8.0±0,1) and proteins elute with a linear concentration gradient of NaCl from 0 to 0.15 M in buffer A. TNF-alpha, suirvey with a column at NaCl concentration Of 0.045-0,07 M, diluted with two volumes of buffer B (10 mm KN2RHO4, pH 8.0±0,1) and put on a hydroxyapatite column (6±2 mg of protein per 1 ml sorbent). The column was washed with buffer B and proteins elute with a linear concentration gradient of potassium phosphate, 0.01 to 0.30 M, pH 8.0±0,1. TNF-alpha, suirvey with a column for the concentrations of potassium-phosphate 0,17-0,24 M, has a purity of 90%. Product cialiswhat against buffer B (10 mm KN2PO4pH 6,7±0,1) and subjected to re-chromatography on hydroxyapatite (load carrier 4±1 mg protein per 1 ml sorbent). The column is washed with buffer and proteins elute with a linear concentration gradient of potassium phosphate, from 0.01 to 0.40 M, pH 6,7±0,1. TNF-alpha, suirvey with a column at a concentration of for 0,19 0,23 M potassium-phosphate, has a purity of 97-100%. The resulting product, containing in the analysis of one protein fraction with a molecular weight of 16.7±0,3 kDa, dialist against buffer D (10 mm KH2PO4pH 7,2±0,1).

Six preparative experiments for selection of recombinant TNF-α summarized in table 1

Table 1
Summary table of experimental data for the preparative separation of recombinant TNF-α man
StageMeasured valueSample preparation, obtained by the proposed method
No. 1No. 2No. 3No. 4No. 5No. 6Average of 6 experiments
The number of used biomass, g
10,0 10,2the 10.1the 10.110,210,0the 10.1
ExtractionTotal proteinmg/ml8,49,77the 17.315,09,95,5
mg6301328,71228,31065,01356,3750
TNF-αpurity, %27203030253327,5
mg170,0265,7368,2319,5339240,0
output %100100100100100100100
Chromatography on DEAE-celluloseTotal proteinmg/ml0,61,022,01,79at 1,8650,45
mg178,8164,2684,0521,8270152,1
TNF-αThe number of the PTA, %60,08948,050,09090,071,2
mg107,3146,14328,3260,9243136,9
output %63,15589,281,771,757,069,6
Chromatography on gap (pH 8.0) and dialyzedTotal proteinmg/ml2,61,076the 3.83,352,12,3
mgto 135.2137,7334,4247,9210140,3
TNF-αpurity, %80,09582,0of 87.09698,089,7
mgto 108.2130, 8mm274,2215,6202137,5
output %63,649,274,567,5to 59.657,362,0
Chromatography on gap (pH of 6.7) and dialysisGeneral b the Lok mg/ml2,151,12,652,842,452,27
mg103,2110260,0194,5198,5140,7
TNF-αpurity, %97,09898,5100,09898,0of 98.2
mg101,1107,8256194,5194,5137,9
output %59,54169,561,057,4of 57.557,6
The release of TNF-α/g biomassthe 10.110,8to 25.319,419,413,816,5

According to the presented data, the average yield of the preparation TNF-alpha is 57.6% of the content in the source cell extracts that are not lower than in the method, the prototype (56% - the data description to the patent 2144958), or of 16.5 mg/g biomass content in the extract 20-30%, which is 1.9 times higher than in the method prototype.

Obtained by the proposed method, the recombinant TNF-alpha man have high electropho micescu purity - 97-100%. After gel electrophoresis in a fully denaturing conditions in the gel with Coomassie blue staining bright blue G250 in perchloric acid in the gel revealed a single band corresponding to the molecular mass 16700 Yes under load 8.3 ág/mm2the surface pocket or 25 μg per well with cross-sectional area of 3 mm2(figa). Staining of proteins by silver nitrate under conditions that allow to identify 0.05 mg in the band (or 1% of impurity proteins in the gel also detected a single band corresponding to TNF-alpha (figb). Load the gel with silver staining is 1.7 ág protein / mm2or 5 μg/well.

The immunoblot of the drug with a monoclonal antibody to TNF-alpha detects no more than 3% dimeric forms of TNF-alpha (figure 3), and some drugs does not detect dimers that meets the requirements for purity of the recombinant protein for medical applications [8, 9].

The content of impurity DNA determined by molecular hybridization with32P-labeled total DNA isolated from strain-producer of TNF-alpha, carried out according to [15], an average of 45 PG/mg protein versus 80 PG/mg protein in the method-prototype (table 2, figure 4).

Certain chemical-enzymatic method, the impurity content of LPS [14] in the preparations of TNF-alpha, obtained by the present method, 5 times the who, than the method-prototype, and an average of 30 ng/mg (versus 160 ng/mg protein in the prototype), see table 2.

The impurity content of proteins producer strain in the final product, as determined by the method of immuno-enzymatic analysis [9], an average of six experiments 111 ng/mg TNF-alpha against 127 ng/mg in the prototype method.

Testing the biological properties of recombinant TNF-alpha man, obtained by the claimed method, conducted on the culture of L929 cells, showed that the drug TNF-alpha has expressed lytic effect on malignant cells, and its specific activity reaches a 3.0×107u/mg protein.

Thus, physico-chemical characterization and biological activity of the final product indicated that recombinant TNF-alpha, obtained by the claimed method, is suitable both for research and for research in medicine.

New compared to the method of the prototype feature is the additional extraction of TNF-alpha from the cell debris by washing the precipitate buffer, raising the pH to 8.0 during the application of the extract on DEAE-cellulose 52, which eliminates loss of the target protein when applied to aminoalkenes and to increase the degree of purification of the product, and two sequential chromatography on hydroxyapatite at different pH (8,0,then 6,7). The combination of these features allows you to obtain highly purified recombinant TNF-alpha man reduced compared with the method of the prototype of the impurity content of bacterial proteins, lipopolysaccharides and DNA at a high yield of the target cytokine.

The use of leaching of cellular debris, obtained after centrifugation destroyed by ultrasound cells, allows for 8-10% increase in the extraction of the recombinant protein, which is advantageous for the scaling process. Increasing the concentration of Tris-HCl 50 mm, pH to 8.0 buffer for the destruction of biomass eliminates the loss of recombinant TNF-alpha in the process of applying to DEAE-cellulose DE 52. Two subsequent phases of the chromatographic purification is performed on the hydroxyapatite. This carrier was synthesized in the laboratory by a known method of Tiselius [16]. The ability to share TNF-alpha and proteins cells of strain-producer of the synthesized media was not inferior costly designs of the firm "BioRad", which eliminates the dependence on imported hydroxyapatite. In addition, the cost of the synthesized hydroxyapatite is many times lower than the cost of imported media.

Thus, the proposed method allows to obtain highly purified recombinant TNF-alpha man with onigen the m content of impurities bacterial LPS and DNA at a high yield of the target cytokine.

The invention is illustrated by the following figures of graphical images.

Figure 1. Electrophoretic analysis of proteins obtained during purification of TNF-alpha, in a 12.5%polyacrylamide gel under fully denaturing conditions.

1 - cell extract;

2 - target fraction with DEAE-cellulose;

3 - target fraction of hydroxyapatite (pH 8.0);

4 - target fraction of hydroxyapatite (pH 6,7).

Figure 2. Electrophoretic analysis of purity of TNF-alpha, selected by the proposed method in a 12.5%polyacrylamide gel under fully denaturing conditions.

And Coomassie blue staining bright blue G250 in perchloric acid.

B - coloring silver.

The numbers below the figure shows the amount of TNF-alpha, introduced into the hole.

Figure 3. The interaction of TNF-alpha monoclonal antibodies to TNF-alpha (immunoblot).

Purified protein was fractionally 12.5%polyacrylamide gel under fully denaturing conditions and were identified by staining Kumasi bright blue G250 in perchloric acid (A) or monoclonal antibodies after the preliminary transfer of proteins from polyacrylamide gel to nitrocellulose membrane (B).

Fig 4. Determination of impurity DNA in four samples of TNF-alpha. Analysis was performed by molecular hybridization with32P-labeled total DNA isolated from strain-ol is ducenta TNF-alpha, on MUK 4.1/4.2.588-96, p.15. In the standard range of DNA took the following amount of DNA on the spot: 2 ng, 0.4 ng, 80 ng, PG 16, 3,2 ng of 0.64 PG 0 PG.

Figure 5. Electrophoretic analysis of proteins extracted from biomass of E. coli SG 20050/pTNF311Δin a 12.5%polyacrylamide gel under fully denaturing conditions.

1 - proteins in the cell extract;

2 - proteins, optionally extracted from the cell debris.

Examples of specific implementation method below.

Example 1. Obtaining highly purified recombinant human TNF-alpha

10 g of E. coli cells SG20050/pTNF311Δ suspended in 70 ml of buffer for destruction (25 mm Tris-HCI, pH 8±0,1, 1 mm EDTA, 1 mm PMSP) to a homogeneous state. Then the suspension was placed in an ice bath and subjected to ultrasonic treatment (installation of UZDN-2T) at a frequency of 22 kHz and the amplitude of 17±2 μm to 5 times for 1 min with an interval of 1 min for cooling of the mixture (the temperature should not exceed 8±2°). Cell extract was separated by centrifugation at 16000 rpm for 50 min (centrifuge J2-21, rotor JA-20, the temperature of 4±2°). For more extraction of TNF-alpha from biomass conduct re-suspension of cell debris in 70 ml of the same buffer, followed by centrifugation as described above. United supernatant applied to a column of DEAE-cellulose DE-52 (volume 140 is l), equilibrated with buffer A (25 mm Tris-HCl, 1 mm EDTA, pH 8±0,1). The column is washed with buffer And the lack of optical density at a wavelength of 280 nm at the exit from the column, then elute proteins with a linear concentration gradient of NaCl from 0 to 0.15 mol/l in buffer A (volume gradient 1300±50 ml). Collect fractions of 10±0.5 ml and analyzed contained proteins by gel electrophoresis in a 12.5%polyacrylamide gel under denaturing conditions on Laemmli [17]. TNF-alpha is usually eluted from the column at NaCl concentrations of 0.045-0.07 mol/liter Fractions containing TNF-alpha are pooled (total volume of 270±30), diluted with buffer B (10 mm KN2PO4pH 8±0,1) and put on a hydroxyapatite column (volume 60 ml), synthesized according to the method of Tiselius and others [16], equilibrated with buffer B. the Column was washed with buffer B until the lack of optical density at a wavelength of 280 nm at the exit from the column, then elute proteins with a linear concentration gradient of potassium phosphate, from 0.01 to 0.30 mol/l, pH 8.0±0,1. Collect fractions of 5 ml and analyzed by electrophoresis as described above. TNF-alpha is usually eluted at a concentration of potassium phosphate 0,17-0,24 mol/L. Faction with the content of TNF-alpha to 80% and more combine (combined volume fractions 90±10 ml) and cialiswhat against 2 l of buffer B (10 mm KN2PO4pH 6,7±0,1) during the night(14-17 hours) at a temperature of +4° C. Then a solution of TNF-alpha put on a hydroxyapatite column (volume 50 ml), equilibrated with buffer C. the Column is washed with buffer to the lack of optical density at a wavelength of 280 nm at the exit from the column, then elute proteins with a linear concentration gradient of potassium phosphate, from 0.01 to 0.4 mol/l, pH 6,7±0,1. Volume gradient of 700 ml, collect the eluate fractions of 4±0,5 ml TNF-alpha is usually eluted at a concentration of potassium phosphate to 0.19-0.23 mol/liter Fractions containing by analysis of one protein fraction with a molecular weight of 16.7±0,3 kDa, unite, transferred to dialysis buffer D (10 mm KN2PO4pH 7,2±0,1) and sterilized by ultrafiltration using a membrane filter with a pore size of 0.22 μm. Electrophoretic analysis of TNF-α stages of purification are presented in figure 1.

The average relative yield TNF-α is 57.6% of its content in crude extract of cells or 165 mg of 10 g of biomass (16,5 mg from 1 g of biomass).

The purity and homogeneity of the preparations of TNF-alpha (substance) was determined by the method of polyacrylamide gel electrophoresis. The drug electrophoretic homogeneous at a load of 8.3 ág/mm2or 25 μg per well when painting Kumasi bright blue G-250 (Fig 2A). When staining with silver nitrate additional bands were not detected when the load on gel 5 μg per well (Fig 2B). The content of m is number of forms of TNF-alpha with a molecular mass of 16.7± 0,3 kDa in reduced conditions when the staining was not less than 97%.

In the absence of the drug TNF-alpha impurities foreign proteins painting immunobloting coincided with electrophoregrams (figure 3).

The specific cytotoxic activity of drugs on cells L929 is (1,85-3,3)X107edict./mg protein. The content of impurity proteins of E.coli is 90-140 ng/mg protein (on average 111 ng/mg), the content of impurities DNA -45 PG/mg of the drug (figure 4) and the LPS content of 30 ng/mg of the drug. Data from six experiments are shown in table 2.

Table 2
Comparative characteristics of quality drugs TNF-alpha, obtained by the proposed method and the method prototype (shown in square brackets are the values of the indicators in the method-prototype)
Of product characteristicsSample preparation, obtained by the proposed method
No. 1No. 2No. 3No. 4No. 5No. 6average
The admixture of proteins in E. coli, ng/mg [127]140,0118110110100,090,0111
The admixture of DNA E. coi, PG/mg [80]80,08040202020,045
The admixture of LPS ng/mg (160)4020402030
Purity (SS G250), % [over 95]97,098,098,5100,098,098,0of 98.2
Purity (color silver), % [not more than 2% impurity proteins)The only band monomerThe only band monomer
Specific activity, u/mg [2×107]3,3×1072,5×1071,85×1072,5×1072,0×1073,0×1072,5×107

From the table 2 data shows that in preparations of TNF-alpha, obtained by the claimed method, the impurity content of cellular proteins in E.coli average of 111 ng/mg protein vs. 127 ng/mg in the method-prototype; the content of impurities DNA - 45 PG/mg of the drug against 80 PG/mg in the method-prototype (1.7 times lower), and the content of FSC 5 times lower than in the prototype (30 ng/mg versus 160 ng/mg).

Example 2. The study of the dependence and the desire TNF-alpha from the secondary extraction cell sediment.

Biomass producer strain suspended in the buffer and treated with ultrasound, as described in example 1. Cell debris was separated by centrifugation, the supernatant is collected and determine its content of protein and the percentage of TNF-alpha. Cellular debris carefully resuspended in the same buffer in a volume of 70 ml, the Suspension is centrifuged in the same conditions as in example 1. In the resulting supernatant determine the protein content and the percentage of TNF-alpha. According to the electrophoretic analysis of the relative content of TNF-alpha in both supernatant is 20-25% (figure 5). Re-extraction allows to increase the yield of recombinant protein in the extraction of 8-10%. Supernatant are combined and passed to the next stage.

Thus, in experiments it was shown that highly purified recombinant TNF-alpha man with low impurities content of bacterial LPS, DNA and partially proteins may be derived from a strain of E. coli SG 20050 transformed by the plasmid pTNF311Δthat encodes a polypeptide TNF-alpha man 5 157 amino acid, by disintegration of cells by ultrasound, Department of cell debris by centrifugation, additional extraction of the target protein by washing the cell debris buffers, three successive stages chromatographic purification extra the aligned proteins on diethylaminoethylcellulose at pH 8.0 and hydroxyapatite at pH 8.0 and then when pH of 6.7. The proposed method is compared with the prototype allows you to:

- to increase by 8-10% extraction of the target protein from the cells of the producer;

to avoid the loss of the target protein when applied to DEAE-cellulose by increasing the pH to 8.0;

to improve the cleaning efficiency on the hydroxyapatite through a chromatography at pH 8.0, and then at pH 6,7;

- to avoid costly imported media - hydroxylapatite and to use instead synthesized in the laboratory by a known method of Tiselius.

While maintaining high product yield and increases the purity of TNF-alpha.

Literature

1. Nedospasov S.A., Turkish R.L., Shakhov A.N. //Results of science and technology. Immunology. 1988. No. 22. S.

2. Cetlinski S.A., Belov, A.A., etc. //Successes of modern biology. 1989. No. 107. Issue 1. S-91.

3. Tracty K.J. //The cytokine Handbook ed. A. Thomson. 1994. Academic. Press. P. 289-304.

4. Kemeny N. Childs Century, Larchian N. et al. //Cancer. 1990. V. 66. # 4. R-663.

5. Takematsu, H., H. Ozawa, T. Yoshimura et al. //British. J. Dermatology. 1991. V.124. N.2. P.209-210.

6. Sheron N, Lau J.Y.N., Daniels H.M et al. //Lancet. 1990. V.336. P.321-322.

7. Patent SU # 1630602, CL 12 N 37/02, publ. BI No. 7 (1991)

8. RF 42-28-9-89. General requirements for medical immunobiological preparations obtained by the method of genetic engineering. Moscow. 1988.

9. EOF 42-226 SU-89. Human recombinant interferon alpha-2 (semi-finished).

10. Nakamura, S., Masegi, S., Fukuoka, M. et al. //Agrical. Biol. hem. 1991. N1. P.53-57.

11. Afanas'ev, V.M., Khlebnikov B.C. //Preparativi. chromatogr. physical asset. substances in polymeric sorbents: proc. Dokl. 1 Uses. proc. Leningrad. 11-13 Oct. 1988 - L. 1988. P.21.

12. Tikhonov W., Yakimov S.A., Korobko VG, Wolfson FN. //Bioorgan. chemistry. 1996. No. 3. Pp.163-167.

13. RF patent №2144958, CL 12 N 15/28, publ. BI No. 3, 2000

14. Denisov L.Y., Baturin I.I., Zakabunin A.I., Afinogenov G.N., Pustoshilova N.M. //IMEI. 1999. No. 5. Pp.109-112.

15. Control methods of medical immunobiological preparations, the input people. HOWTO MUK 4.1/4.2.588-96. The Ministry Of Health Of Russia. M. 1998.

16. Tiselius, A., Hjerten, S., Levine O. // Arch Biochem. Biophys. 1956. V. 65. P. 132.

17. Laemmli U.K. //Nature (London). 1970. V. 227. R. 680-685.

A method of obtaining a purified recombinant polypeptide with properties of tumor necrosis factor alpha person, including the cultivation of the producer strain E. coli SG 20050/pTNF311Δthe destruction of cells by ultrasound, removing cellular debris and chromatography on columns with sorbent, wherein the target protein is additionally removed by washing the cell debris buffer for cell disruption, and the chromatographic purification of the product is carried out sequentially on DEAE-cellulose in a linear gradient of NaCl at pH 8.0±0.1 and twice on hydroxyapatite in a linear gradient of phosphate, at pH 8.0±0,1 and then at pH 6,7±0,1.



 

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5 cl, 1 dwg, 1 ex

FIELD: bioengineering; genetic engineering; medicine; methods of production casamino acids.

SUBSTANCE: the invention is pertaining to the field of bioengineering, genetic engineering, medicine, in particular, to the methods of production of components for nutrient mediums from hydrolysates of animal protein. The invention offers the method of production of casamino acids by the method of the gel permeation chromatography of the hydrolyzed crude acid casein with the contents of the general nitrogen - 0.7-0.95 g in 100 ml of the solution and concentration - 6-10 % on Sefadex G-15, eluating by a distilled water of fractions of an eluate, selection of the active fractions of an eluate by a spectophotometery of portions of the eluate (D254), evaporation of the active fractions under vacuum at the temperature of no more than 55°C. The method allows to simplify the process of production of casamino acids, to reduce its cost and also to obtain casamino acids possessing the high growth- stimulating activity.

EFFECT: the invention ensures simplification of the process of production of casamino acids, reduction of its cost and also production of casamino acids possessing the high growth- stimulating activity.

2 cl, 2 dwg, 1 tbl, 1 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.

EFFECT: improved preparing method, valuable biological and biochemical properties of system.

49 cl, 5 dwg, 2 tbl, 9 ex

The invention relates to medicine and relates to a derivative of annexin with endogenous chelation sites and which can be used in the diagnosis of thrombosis
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