Strain of bacteria escherichia coli jm109/phins21 - producer of hybrid protein with human proinsulin and method for production of human proinsulin

FIELD: medicine.

SUBSTANCE: recombinant strain Escherichia coli is produced, which contains plasmid pHINS21 (Escherichia coli JM109/ pHINS21), defining synthesis of hybrid protein, made of N-terminal fragment of human gamma-interferon and human proinsulin, joined by peptide linker, which contains site of splitting with enterokinase (Asp4Lys). Yield of hybrid product that includes proinsulin, provided with new strain-producer, makes at least 30% of total amount of cell protein. Method is suggested for preparation of human proinsulin, including cultivation of strain-producer Escherichia coli JM109/pHINS21, separation of inclusion bodies and their dissolution, renaturation of hybrid protein and its cleaning with ion-exchange chromatography, splitting of hybrid protein with enterokinase or its catalytic subunit and cleaning of proinsulin by ion-exchange chromatography on sorbates with sulfprofile groups.

EFFECT: invention simplifies technological process for production of recombinant human proinsulin and improves conditions of its execution from the point of view of safety engineering.

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The invention relates to the field of biotechnology, namely the preparation of recombinant human proinsulin, and can be used in medicine.

Proinsulin is synthesized in the rough endoplasmic reticulum b-cells of the islets of Langerhans of the pancreas in the form of its predecessor preproinsulin (molecular weight 11500 Yes). A leader sequence consisting of 23 amino acid residues, directs the molecule, the precursor in the Golgi apparatus and there is cleaved. The result is the proinsulin molecule (molecular weight of about 9000 Yes), the receiving conformation necessary for the proper formation of disulfide bridges. Then proinsulin is cleaved in a few specific areas with the formation of Mature insulin and C-peptide, which is deposited in secretory granules.

Recent medical studies show that proinsulin may block the destruction of b-cells of the pancreas, acting as local protector as well as to stimulate their regeneration. Therefore, the development of methods for obtaining recombinant human proinsulin for the prevention and treatment of diabetes is extremely important.

A method of obtaining recombinant human proinsulin [1-3], which includes the lysis of cellsE. coliexpressing hibri the major proteins with human proinsulin in the form of Taurus include, dissolution Taurus inclusion in a buffer solution containing urea or guanidine chloride, sulfonation of the hybrid proteins of the sulfonate sodium and tetrathionate sodium, centrifugation hybrid protein-S-sulfonate of proinsulin, the separation of S-sulfonate of proinsulin from the leader peptide by treatment with methyl zian, clean S-sulfonate of proinsulin anion-exchange chromatography, re-laying of the chain S-sulfonate of proinsulin and purification of proinsulin human adsorption chromatography.

The disadvantages of the described method is a multi-stage process and the use of the cleavage of a hybrid protein is highly toxic reagent methyl cyanide.

Therefore, at present there is a need to develop improved methods for obtaining recombinant human proinsulin, which would be high-performance and non-toxic.

To obtain recombinant proteins of the hybrid polypeptides of known approaches, in which cleavage of the final product is performed using a highly specific proteolytic enzymes. Such methods include the construction of hybrid proteins containing the site of recognition of specific proteases between a protein carrier (leader peptide) and a target protein, the purification of the synthesized gibridnogo the protein and its enzymatic cleavage. One of the highly specific proteases used for this purpose is enterokinase (enteropeptidase, KF 3.4.21.9), which cleaves the peptide bond after the sequence (Asp)4Lys.

Description of the invention

The present invention relates to a hybrid protein is the precursor of human proinsulin sequence presented in figure 2, comprising the amino acid sequence of the leader peptide representing the N-terminal fragment of IFN-γ of human (SEQ ID NO:1), peptide linker (SEQ ID NO:2), containing the cleavage site of enterokinase and human proinsulin (SEQ ID NO:3).

In addition, the invention relates to DNA encoding specified hybrid protein, the nucleotide sequence presented in figure 2.

The invention relates to a recombinant plasmid containing the indicated DNA that encodes the specified hybrid protein with human proinsulin, and also includes regulatory elements. As regulatory elements and genetic markers that are used in the plasmids of the present invention, can be illustrated by the following non-limiting elements and markers: tac-promoter transcription terminator of transcription of ribosomal operonE. coligene β-lactamase (bla), used as a genetic marker that defines the stability of t is informirovannii the plasmid pHINS21 cells of bacteria to ampicillin.

Specifically, the invention relates to recombinant plasmid pHINS21 (figure 1)containing the specified DNA encoding a hybrid protein (figure 2), in which the amino acid sequence of the leader peptide representing the N-terminal fragment of IFN-γ human, and human proinsulin are connected by a peptide linker containing the cleavage site of enterokinase, with the indicated plasmid has a molecular weight of 2.36 MDA and includes a unique recognition sites of the restriction endonucleases, located on the following distance to the right of the website RI: BamHI - 159 BP, HpaI - 185 BP, HindIII - 442 BP, PvuI - 1382 BP

New hybrid protein provides a high yield of the target product of proinsulin person through effective renaturation and efficient cleavage by enterokinase specified hybrid protein.

The invention relates to transformed cellsEscherichia colicontaining this recombinant plasmid, producing a hybrid protein of human proinsulin. Specifically, the invention relates to a strain of bacteriaEscherichia coliJM109/pHINS21 producer of the hybrid protein of human proinsulin.

In addition, the claimed hybrid protein with human proinsulin, for example, produced by a strain ofE. coliJM109/pHINS21, can be used in the method of producing human insulin. The new g is Britny protein transform into insulin by enzymatic cleavage of the specified hybrid protein enterokinase, trypsin and carboxypeptidase B.

The invention relates to a new method of producing human proinsulin. The inventive method of producing human proinsulin includes the cultivation of the strain-producerEscherichia colidestruction of bacterial cells, the selection Taurus inclusion, their dissolution in buffer containing urea and dithiothreitol, renaturation and cleaning denaturirovannogo hybrid protein with human proinsulin, its cleavage with a specific protease, followed by purification and obtaining the target product. In the particular case, the method includes cultivating a new strain ofE. coliJM109/pHINS21 carrying a new plasmid pHINS21, cleavage of the hybrid protein can be carried enterokinase (or its catalytic subunit), and obtained after enzymatic cleavage of proinsulin purified by ion exchange chromatography on SP-sepharose with subsequent high-performance liquid chromatography on reversed phase.

An advantage of the claimed method of producing human proinsulin is a simplification of the process and exclusion from technology highly toxic reagent.

Detailed description of the invention

Created a new recombinant plasmid pHINS21, which determines the synthesis of a hybrid protein with molecular weight of about 15,85 kDa, in which the amino acid posledovatel the activity leader peptide, representing the N-terminal fragment of IFN-γ human, and human proinsulin are connected by a peptide linker containing the cleavage site of enterokinase. New hybrid protein whose properties determine its effective renaturation and efficient enzymatic cleavage by enterokinase, provides a high yield of the target product of proinsulin person.

The optimal length of the linker and its amino acid composition were determined experimentally.

Recombinant plasmid pHINS21 designed based on the known plasmids pHINS05 [4]. DNA plasmids pHINS05 was subjected to complete hydrolysis by restrictase BclI and HpaI, and the resulting fragment BclI-HpaI (4,2 KBP) ligated with oligonucleotide duplex obtained by annealing synthetic oligonucleotides: sense represented in the sequence SEQ ID NO:4, and antisense, represented in the sequence SEQ ID NO:5. As a result, in the gene of the hybrid protein to the DNA of human proinsulin integrated nucleotide sequence encoding the cleavage site of enterokinase, namely peptide fragment (Asp)4Lys.

Legirovannoi mixture used to transform competent cellsE. coliJM109. The result obtained recombinant plasmid pHINS20, the structure of which was confirmed by restriction analysis. The structure of the s (2), encoding a hybrid protein, was confirmed by sequencing.

To increase the number of copies obtained plasmids pHINS20 in the bacterial cell it was deleterule on rop-gene (negative control copy number) [5]. With this purpose, DNA plasmids pHINS20 were subjected to complete hydrolysis by restrictase Eco47III and SnaI, and the resulting fragment Eco47III-SnaI (a 3.6 KBP) ligated. The result obtained recombinant plasmid pHINS21, the structure of which was confirmed by restriction analysis and sequencing.

The new recombinant plasmid pHINS21 encodes a hybrid protein size is 143 amino acids with a molecular mass of 15,85 kDa, in which the amino acid sequence of the leader peptide representing the N-terminal fragment of IFN-γ of human (SEQ ID NO:1), and human proinsulin (SEQ ID NO:3) are connected by a peptide linker (SEQ ID NO:2), containing the cleavage site of enterokinase.

The indicated plasmid consists of a fragment of the BamHI-EcoRI plasmid RCC-3 [6], containing the promoter transcription tac; fragment EcoRI-HindIII, including hybrid protein gene with the nucleotide sequence presented in figure 2, encoding the N-terminal fragment of IFN-γ person, a peptide linker with the site of cleavage of enterokinase and amino acid sequence of human proinsulin; fragment HindIII-SnaI plasmids RCC-3, containing the terminator of transcr is PCIe ribosomal operon E. coligene β-lactamase (bla), the site of initiation of replication (ori); and Eco47III-EheI fragment of plasmid RCC-3; a unique recognition sites of restriction endonucleases that are located in the following distance to the right of the website RI: BamHI - 159 BP, HpaI - 185 BP, HindIII - 442 BP, PvuI - 1382 BP

Producing strains ofE. coliJM109/pHINS21 obtained by transformation of cellsE. colistrain JM109 the plasmid pHINS21. After transformation of the selected colonies grown on the medium with ampicillin, perhaps, plasmids, and are subjected to restriction analysis and sequencing. The cell line that carries a plasmid pHINS21, repeatedly subcultured on a medium with agar with addition of ampicillin and received monoclonal culture inoculant 5 ml of liquid medium with ampicillin. The culture is tested for the presence of induced expression of the hybrid protein, Packed, add glycerol and stored at minus 40°C.

A new strain ofEscherichia coliJM109/pHINS21 carrying plasmid pHINS21, is the producer of the hybrid protein containing the amino acid sequence of human proinsulin, and is characterized by the following features.

Morphological features: the cells are small, rod-shaped, gram-negative, risperadone size C,5 μm, mobile, with distinct cells activate after the induction of the synthesis of a hybrid protein.

Cultural characteristics: the growth of nagaretani environment LB colonies are round, smooth, translucent, shiny, grey. The edge is smooth, the diameter of the colonies 1-3 mm thick, pasty consistency. Growth in liquid media (LB, minimal medium with glucose) is characterized by smooth blushing.

Physiological and biochemical characteristics: the cells grow at temperatures 4-42°C, the optimum pH of 6.8 to 7.6. As the source of nitrogen used as mineral ammonium salts, and organic compounds: amino acids, peptone, tripton, yeast extract. As a carbon source during growth on minimal medium using glycerol, carbohydrates, amino acids.

Resistance to antibiotics: cell producer strain are resistant to ampicillin (500 mg/ml), due to the presence of plasmid gene of β-lactamase (bla).

The stability of the plasmid in the strain. While maintaining the cells in a few months on agar LB medium containing ampicillin, there is not loss or rearrangement of plasmid influencing the expression of a hybrid protein.

B new strain of hybrid protein after induced expression accumulates in the form of Taurus inclusion, and its content is at least 30% of the total protein of the cell.

Obtained producing strains ofE. coliJM109/pHINS21 deposited in the collection of microorganisms of JSC "National Biotechnology" number 21-06.

The new recombinant plasmid pHINS21 provides effective the first hybrid protein biosynthesis, containing human proinsulin, and other bacterial strains ofEscherichia colifor example in the strain-hostE. coliBL21 as described below.

As a non-limiting example in the description of the method of producing human proinsulin usingE. coliJM109/pHINS21. The cultivation conditions of the producer strain and selection Taurus inclusion are not determinative, and the person skilled in the art on the basis of General knowledge will select similar conditions that do not affect the achievement of the technical result.

The growing culture of strain-producerE. coliJM109/pHINS21 spend on a nutrient medium on the basis of casein hydrolysate and yeast extractSaccharomyces cerevisiae. For the induction of the synthesis of a hybrid protein in the middle of the logarithmic phase of growth bring 1-isopropyl-β-D-1-thiogalactopyranoside (IPTG). The cultivation is continued until formation of the desired level of intracellular inclusions hybrid protein (Taurus enable). After growing the culture precipitated cells destroy one of the acceptable methods and calf enable separated by centrifugation. Sediment Taurus inclusion (paste) contains about 5-7% of the hybrid protein of proinsulin.

The selection of the hybrid protein of Taurus include, in its enzymatic cleavage and purification of proinsulin carried out according to the following scheme:

• Taurus enable RA is tworay in 0,1M buffer Tris-HCl, pH 8.0, containing 8 M urea, followed by the addition of dithiothreitol to a final concentration of 10 mm;

• hybrid protein centurybut in 10-fold volume of 0.1 m glycine buffer, pH 9-11 at a temperature of 10-14°C for 20-24 h;

• cleaning denaturirovannogo hybrid protein is carried out by chromatography on DEAE-sepharose FF, balanced 0,05M Tris-Hcl buffer, pH 7.0 and 7.5, containing 0,15M NaCl. Protein elute with a linear gradient of 0.15-0,4M sodium chloride in equilibrating buffer;

• purified hybrid protein break down enterokinase (or its catalytic subunit) within 12 to 24 hours at pH 7.0-7.5 and a temperature of 16 to 26°C. the Reaction is stopped podkisst hydrolyzed with hydrochloric acid to a pH of 4.6 to 5.0, and the formed precipitate of impurity proteins separated by centrifugation;

• proinsulin purified by ion exchange chromatography on SP-sepharose FF in 0,05-0,2M ammonium acetate buffer, pH 3.0 to 6.0, containing 1-2M urea. Adsorbed protein elute with a linear gradient of KCl from 0 to 0.5m in equilibrating buffer;

• further purification of proinsulin carried out by the method of reversed-phase high-performance liquid chromatography (RP HPLC).

Brief description of figures

Figure 1 shows the physical map of recombinant plasmid pHINS21.

Figure 2 presents the nucleotide sequence of the gene of hibri the nogo protein prosolia member plasmids pHINS21 and coded them amino acid sequence.

Figure 3 shows HPLC analysis of the material obtained after purification denaturirovannogo hybrid protein with human proinsulin on DEAE-sepharose.

Figure 4 shows HPLC analysis of the products of the cleavage of the hybrid protein with human proinsulin enterokinase.

Examples

The invention is illustrated by the following not limiting examples.

Example 1.Construction of plasmids pHINS21

Plasmid pHINS21 design based on the known plasmids pHINS05 [4]. Plasmid DNA pHINS05 subjected to complete hydrolysis by restrictase BclI and HpaI. For this purpose, 5 μg of plasmid DNA HINS05 in 20 μl of a buffer containing 33 mm Tris-acetate, pH of 7.9, 66 mm K-acetate, 10 mm Mg-acetate and 0.1 mg/ml BSA (buffer 1) and 10 UNITS of restrictase BclI and HpaI incubated for 1.0 hour at 37°C. From the resulting hydrolysate produce DNA fragment BclI-HpaI size of about 4.2 KBP using electrophoresis in 0.8% of the gel-melting agarose. Further DNA deproteinizing phenol, a mixture of phenol and chloroform (1:1), chloroform, precipitated with ethanol, dissolved in 20 μl of water. The resulting fragment BclI-HpaI, containing the gene of the hybrid protein and the vector portion of the plasmid HINS05, are ligated with 20-fold molar excess of oligonucleotide duplex obtained by annealing synthetic oligonucleotides: sense represented in the sequence SEQ ID NO:4, and antisense, pre is raised in the sequence of SEQ ID NO:5.

As a result, in the gene of the hybrid protein to the DNA of human proinsulin integrated nucleotide sequence encoding the cleavage site of enterokinase.

Competent cells of strainE. coliJM109 transformed legirovannoi mixture (10 μl) and plated on LB-agar containing 100 μg/ml ampicillin. From grown colonies produce plasmid DNA is sequenced and between sites restricts EcoRI and HpaI to confirm the insertion in the linker part of the gene of the hybrid protein. The result is the plasmid HINS20.

To obtain plasmids with deletions on rop-gene (negative control copy number) to 5 μg of plasmid DNA HINS20 in 20 μl of buffer 1 add 10 UNITS of restrictase Eco47III and SnaI, generating ”blunt” ends, and the mixture is incubated for 1 hour at 37°C. Next, the DNA deproteinizing phenol, a mixture of phenol and chloroform (1:1), chloroform, precipitated with ethanol, dissolved in 20 μl of water. Thus obtained DNA are ligated in 30 μl of buffer for ligation in the presence of 5 Units of DNA ligase of phage T4 for 16 h at 8°C. Legirovannoi mixture (10 ál) transform competent cells of strainE. coliJM109 and plated on LB-agar containing 100 μg/ml ampicillin. Selected bacterial clones carrying the plasmid DNA by $ 3.6 KBP Selected plasmid is subjected to restriction analysis and is sequenced by the method of Singer. The result PLA the Ministry of foreign Affairs pHINS21.

Example 2.Obtaining strain ofE. coliJM109/pHINS21 and determine the level of productivity

The plasmid pHINS21 transform competent cells of strainE. coliJM109 and plated on LB-agar containing 100 μg/ml ampicillin. Separately localized colony subcultured three times on plates with LB-agar containing 100 μg/ml ampicillin. Received monoclonal culture inoculant 5 ml liquid LB medium with ampicillin and incubated overnight with vigorous shaking at 37°C.

Obtained producing strains ofE. coliJM109/pHINS21 stored in 20% glycerol at minus 40°C.

To determine the level of induced expression of the hybrid protein overnight culture inoculated at a dilution of 1:50 in 5 ml liquid LB medium containing 100 μg/ml of ampicillin, and cultivated until the turbidity of 0.8 at 37°C on a shaker at 200 rpm To culture add IPTG to a concentration of 1.0 mm and continue incubation in the same conditions for 3 hours. Cells are harvested by centrifugation, the precipitate is suspended in a buffer containing 62.5 mm Tris-HCl, pH 6.8, 3% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 10% glycerol and 0.01% bromophenol blue and heated for 3 min in a boiling water bath. The obtained cell lysate analyzed by electrophoresis in 18% polyacrylamide gel with sodium dodecyl sulfate [7]. Gel stain Coomassie R-250, scan and spend it densitometry. According to densitometry with the holding of the hybrid protein is at least 30% of the total protein of the cell.

Example 3.Obtaining strain ofE. coliBL21/pHINS21 and determine the level of productivity

Competent cells of strainE. coliBL21 transformed plasmid DNA pHINS21 and plated on LB-agar containing 100 μg/ml ampicillin. Separately localized colony subcultured three times on plates with LB-agar containing 100 μg/ml ampicillin. Received monoclonal culture inoculant 5 ml liquid LB medium and incubated overnight with vigorous shaking at 37°C.

Obtained producing strains ofE. coliBL21/pHINS21 stored in 20% glycerol at minus 40°C.

Determination of productivity of the strain ofE. coliBL21/pHINS21 spend the same method as for strain,E. coliJM109/pHINS21 described in example 2. According to densitometry content of the hybrid protein in induced cells obtained strain is not less than 30% of the total protein of the cell.

Example 4.Getting proinsulin person when using the strain-producerE. coliJM109/pHINS21

Step 1.Selection Taurus inclusion containing a hybrid protein with human proinsulin

Cultivated strain ofE. coliJM109/pHINS21 the nutrient medium on the basis of the hydrolysate of casein and Baker's yeast extract. The induction of the biosynthesis of the hybrid protein is performed by making the inductor IPTG in the middle of the logarithmic phase of growth culture. Cultivation continued the t before the formation of intracellular inclusions of the hybrid protein. After growing the culture precipitated and used for selection Taurus inclusion containing a hybrid protein of proinsulin.

Cells are suspended in buffer a containing 50 mm sodium phosphate (disubstituted), 1 mm EDTA, 0.2m sodium chloride per 1 g of biomass 5-10 ml of buffer and destroy on the cage Gaulin. The destroyed cells are centrifuged for 20 min on a Beckman J-30I at 8000 Rev/min the precipitate resuspended in buffer a containing 1% Triton X-100, incubated for 1 hour and the suspension is centrifuged for 20 min on a Beckman J-30I at 8000 Rev/min the Precipitate re-resuspended in the buffer and precipitated in a centrifuge. The washed precipitate Taurus inclusion (paste)containing about 5% of the hybrid protein of proinsulin, Packed, frozen and stored at minus 40°C.

Stage 2.Resaturate and the selection of the hybrid protein

5 g of thawed paste Taurus include dissolved in 100 ml of buffer solution containing 0.1m Tris-HCl, pH 8.0, 10 mm dithiothreitol and 8M urea, and incubated with stirring for 18-24 h at 14°C. the Solution is centrifuged for 20 min on a Beckman J-30I at a speed of 8000 rpm at 14°C. Renaturation hybrid protein contained in the supernatant, perform 10-fold dilution in 0.1m glycine buffer, pH 9-11. The hybrid protein solution incubated for 20-24 hours, stirring and maintaining the temperature of 10-14°C. The process of renaturation of hybrid protein is controlled by the method OF HPLC. After the formation of the correctly closed S-S bonds in 50-70% of the hybrid molecules of the protein solution was acidified with 2 N. hydrochloric acid to pH 7.0-7.2 and the formed precipitate was separated by centrifugation.

Content denaturirovannogo hybrid protein in the supernatant is 112 mg

The solution of the hybrid protein applied to the column volume of 100 ml, filled with DEAE-separate FF, pre-equilibrated 0,05M Tris-Hcl buffer, pH 7.0 and 7.5. The column was washed with equilibrating buffer containing 0,15M NaCl. Adsorbed protein elute with a linear gradient of 0.15-0,4M sodium chloride in equilibrating buffer. The fractions containing the hybrid protein, analyzed by RP HPLC method (figure 3). The result is 95 mg of purified hybrid protein.

The compliance of the resulting hybrid protein with human proinsulin was confirmed by the method of mass spectrometry according to the coincidence with the calculated molecular weight of the specified protein.

Step 3.Enzymatic cleavage of the hybrid protein

The enzymatic hydrolysis reaction is carried out at a temperature of 25°C with constant stirring. To the solution of purified hybrid protein add CaCl2up to a concentration of 1 mm. Then the reaction mixture is make a solution of recombinant enterokinase (catalytic subunit enterocin the s ox) (Novagen) at the rate of 1 IU 4 mg protein and incubated for 20 hours. The products of hydrolysis of the hybrid protein analyzed by the method OF HPLC. 4 shows HPLC analysis of the products of fission hybrid protein enterokinase after 20 h of incubation. The cleavage reaction is stopped podkisst material 2 N. hydrochloric acid to pH 4.0 to 5.0. The precipitate of impurity proteins separated by centrifugation.

Step 4.Purification of proinsulin human

The clarified solution containing proinsulin, is applied to the chromatographic column with a volume of 20 ml, filled with SP-separate FF, pre-equilibrated 0,03M ammonium acetate buffer, pH 5.0, with 2M urea. The column was washed with equilibrating buffer until reaching the baseline control flow densitometer. The elution of sorbed proinsulin spend a linear gradient of KCl from 0 to 0.5m in equilibrating buffer. The content and purity of human proinsulin in the collected fractions determined by the method OF HPLC. Combined fractions containing 54 mg of proinsulin person with a purity of at least 85%.

Further purification of proinsulin carried out by the method OF HPLC on a preparative chromatograph ”Armen” (France). Column volume of 200 ml, filled with Kromasil C18, balance 20%isopropanol with 0.1% triperoxonane acid and submit a solution of proinsulin from the previous stage cleaning with 54 mg of protein. Protein e is wirhout gradient of isopropanol (0.1% triperoxonane acid) from 20 to 40%. The collected fractions of the main peak of proinsulin to analyze the content of impurities by the method OF HPLC.

After cleaning they receive 46 mg purified human proinsulin content of the main substance of 98%.

The authenticity of the obtained target product was confirmed by matching the calculated molecular mass of human proinsulin with the molecular weight of the target product, a specific method of mass spectrometry.

Example 5

Obtained in example 4 (step 2) a hybrid protein with human proinsulin were digested with enterokinase from a natural source.

The reaction of the enzymatic cleavage of the hybrid protein is carried out at a temperature of 25°C with constant stirring. To the solution of purified hybrid protein add CaCl2up to a concentration of 1 mm. Then the reaction mixture is make the solution enterokinase (Sigma, USA) at a rate of 1 IU 4 mg protein and incubated for 16 hours. The products of hydrolysis of the hybrid protein analyzed by the method OF HPLC. The cleavage reaction is stopped podkisst material 2 N. hydrochloric acid to pH 4.0 to 5.0.

Sources of information

1. RF patent 2203949. A method of producing human proinsulin.

2. U.S. patent 5952461. Process for preparing human proinsulin.

3. European patent EP1042479. A process for preparing human proinsulin.

4. RF patent 2263147 C1, 27.10.2005 St. Bull. No. 30.

5. Twigg A.J. and Sherrat D.//Nature, 1980, v.283, p.216-218.

6. Brosus J., Dull T.J., Sleeter D.D., H.F. Noller//J. Mol. Biol., 1981, v.148, p.107-127.

7. Laemmli U.K.//Nature, 1970, v.227, p.680-687.

1. The bacterial strain Escherichia coli JM109/pHINS21 producing a hybrid protein containing human proinsulin.

2. A method of producing human proinsulin, including
the cultivation of the producer strain according to claim 1,
destruction of bacterial cells,
selection Taurus inclusion, their dissolution in buffer containing urea and dithiothreitol,
renaturation and cleaning denaturirovannogo hybrid protein with human proinsulin,
cleavage of the hybrid protein by enterokinase or its catalytic subunit,
cleaning and obtaining the target product.

3. The method according to claim 2, characterized in that
obtained after enzymatic cleavage of proinsulin purified by ion-exchange chromatography sorbents with sulfopropyl groups.

4. The method according to claim 3, characterized in that
after enzymatic cleavage of the hybrid protein purification of proinsulin carried out by chromatography on SP-sepharose FF in the 0.05-0.2m ammonium acetate buffer, pH 3.0 to 6.0, containing 1-2M urea, and adsorbed protein elute with a linear gradient of KCl from 0 to 0.5m in equilibrating buffer.



 

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6 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.

EFFECT: invention provides for preparation of single type of antibodies, which controls activity of NK cells of various type, provides for amplification of their cytotoxicity, which may find application in therapy, for increase of activity or cytotoxicity of NK cells in individuals without preliminary detection of HLA type in individual.

7 cl, 13 dwg, 4 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: there is offered application of humanised fused protein for making a medicine used for stimulation of immune response and stabilisation of disease progressing in patients with GD2-positive tumours. The antibody contains antibody H14.18 caught with surface glycosphingolipid GD2 of human cells, and cytokine IL2. There is disclosed method of increase in ADCC and lysis activity of natural killers in cancer patients by introduction of the fused protein. The invention can be applied in GD2-overexpression cancer therapy.

EFFECT: application of the invention provides low-immunogenicity antibody.

2 cl, 8 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.

EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.

4 cl, 6 dwg, 22 tbl, 19 ex

FIELD: chemistry, medicine.

SUBSTANCE: claimed is novel hybrid protein CFP10-ESAT6 from M. tuberculosis, inducing reaction of hypersensitivity of delayed type with respect to M. tuberculosis. Chimeric NA, coding claimed protein is described. Described is method of obtaining claimed protein by cultivating cells of strain BL21(DE3) E.Coli, transformed with constructed recombinant expression vector on the basis of plasmid pET22b(+). Claimed is dosed medicinal form, containing claimed protein, for intracutaneous injection for diagnostics of tuberculosis infection.

EFFECT: high output of protein CFP10-ESAT6, which possesses specific immunogenicity.

8 cl, 2 dwg, 7 tbl, 3 ex

FIELD: genetic engineering.

SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.

EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.

9 cl, 6 ex

FIELD: medicine.

SUBSTANCE: polypeptides include single-domain antibody against vWF, A1 domain of vWF, A1 domain of activated vWF, A3 domain of vWF, gp1b and/or collagen. Invention claims methods of obtaining indicated polypeptides, methods of coating devices applied in medical practice (e.g. in X-ray structural analysis, endoprosthetics) with indicated polypeptides.

EFFECT: obtainment of polypeptides for treatment of diseases requiring modulation of thrombocyte-mediated aggregation.

40 cl, 69 ex, 30 dwg, 32 tbl

FIELD: pharmacology.

SUBSTANCE: claimed invention relates to field of biotechnology and immunology. Described is physiologically active protein conjugate. Protein conjugate includes physiologically active polypeptide, which is covalently connected with Fc fragment of immunoglobulin by means of polyethylene glycol. Described is method of obtaining protein conjugate. It can find application in production of various polypeptide medications of prolonged action.

EFFECT: increased physiological activity in vivo construction in comparison with native physiologically active polypeptide and increase of half-life in serum of physically active polypeptide with minimal risk of inducing undesirable immune response.

17 cl, 18 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: method of augmentation of duration of action of physiologically active polypeptide in vivo is described. Physiologically active polypeptide is conjugated with Fc fragment of immunoglobulin by means of PEG. Invention use, in comparison with native physiologically active polypeptide, provides the raised physiological activity in vivo designs and-or augmentation of time of a semilife in Serum of physiologically active polypeptide with the minimum risk of induction of undesirable immune responses.

EFFECT: possibility of application of bond at manufacturing of various polypeptides medicinal preparations of the prolonged action.

11 cl, 18 dwg, 8 ex

FIELD: medicine; pharmacology.

SUBSTANCE: immunogenic hybrid polypeptide includes mimetic peptide of V-cellular epitope of apolypoprotein B-100 in which C-end of mimetic peptide is merged with N-end of T-helper epitope. Amino acid sequences of polypeptide variants are presented in description. Described is method of specified polypeptide production providing application of host cell transformed with recombinant express vector including gene coding specified polypeptide. Besides, invention concerns vaccine composition including specified immunogenic hybrid polypeptide for obesity prevention or treatment, recombinant express vector and host cell.

EFFECT: excellent anti-obesity activity without induction of immune response or severe by-effects.

15 cl, 25 dwg, 4 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

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