The enzymatic method of obtaining basic fibroblast growth factor bfgf (10 - 155) and a pharmaceutical composition based on it

 

(57) Abstract:

Usage: biotechnology, pharmacology. The inventive basic fibroblast growth factor bFGF (10 - 155) is produced by forming a complex with the interaction of the protective agent is heparin or heparan sulfate with bFGF (2 - 155) and/or bFGF (3 - 155) and subsequent enzymatic processing of complex pepsin or a cathepsin D. Then bFGF (10 - 155) release from the complex with a protective agent. 2 S. and 3 C.p. f-crystals.

The present invention relates to a method of enzymatic obtain basic fibroblast growth factor bFGF, as well as to pharmaceutical compositions based on it.

Factor bFGF was originally isolated from brain and pituitary in the form of the polypeptide of 146 amino acids (Esch and other PNAS, USA, 82, 6507-6511, 1985). Was cloned gene bFGF bull (Abraham and others, Science, 233, 545 548, 1986). The nucleotide sequence was determined 155-amino acid bFGF-product broadcast. Further research showed that 154-amino acid bFGF can be removed along with the 146-amino acid bFGF from normal tissue of the pituitary adding enzyme inhibitors (Ueno and others Biochem. Biophys. Res. Comm. 138, 580 -588, 1986), and that the acid protease in the brain and hepatoma cells break down bFGF (Klagsbrum and other PNAS U is broblasts for therapeutic use. However, after the expression derived growth factors could be processed only in the mixture of their different forms. FGF-factors are no exception in this respect (Barr and others, J. Biol. Chem. 263, 31, 16471 16478, 1988).

The authors invented a method of obtaining bFGF factor, truncated at its N end. This method can be used to obtain a 146-amino acid forms of bFGF from its longer forms to obtain the only forms of bFGF from a mixture of different BFGF.

Accordingly, the present invention provides a method of enzymatic obtain basic fibroblast growth factor bFGF (10 155), including the interaction of bFGF with a protective agent with a ratio of protective agent: BFGF from 0.5 to 1 1 to 10 (weight/weight), the formation of the complex between the protective agent and bFGF, and processing the received complex proteolytic enzyme, and as bFGF use bFGF (2 155) and/or bFGF (3 155), as a protective agent use heparin or heparan-sulfate, and the resulting complex is treated with pepsin or a cathepsin D, followed by separation of the target product.

The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a major factor in the company fibril lnai length is 155 amino acid residues and can be designated as the 155-BFGF or (1 155) bFGF. The invention can be used for a mixture of bFGF from the 154 amino acid residues ((2 155) bFGF and bFGF of 153 amino acid residues ((3 155)bFGF).

BFGF or a mixture of different bFGF is usually obtained using the techniques of recombinant DNA. Various forms of bFGF receive in the form of such mixtures by treatment of the product broadcast on its N end. One or each bFGF may be human, murine or bFGF rodents.

The adduct can be formed between the protecting agent for bFGF, which are selected from heparin or heparan sulfate, and a mixture of bFGF in any convenient form. Protecting agent and bFGF usually give in aqueous solution. This solution may contain a buffer.

There might also be an antioxidant, such as dithiotreitol, to prevent oxidation of the protein. The ratio of bFGF protecting agent is from 0.5 to 1 1 to 10 (weight/weight), such as from 1 1 to 5 1 (weight/weight). Protection of bFGF in the form of adducts prevents further hydrolysis adding pepsin A.

Pepsin A (EC 3.4.23.1) or cathepsin D (EC 3.4.23.5) is then in contact with the adduct. This leads to the specific cleavage 9 10 Leu-Pro communication bFGF contained in the adducts. The enzyme can be provided in a solution of the adduct.

Therefore, the enzyme may be added to races which is within, for example, in the case of pepsin A, from 30 minutes to 10 hours, for example from 1 to 8 hours Incubation generally longer than for cathepsin D, for example from 90 to 130 h is appropriate interval of about 110 hours, the Temperature of incubation can be from 5oC to room temperature, for example about 10oC. the Incubation is carried out until when the reaction is complete.

On the contrary, protecting agent can be immobilized on the substrate with the formation of the affinity column. Can be used with any suitable substrate, such as an agarose gel or beads sewn dextranomer gel (for example, Sepharose). The bFGF solution passed through the column. bFGF contact with the protecting agent and are retained on the column. The enzyme solution may then be passed through the column. Finally, a shortened one kind of bFGF can be blueraven from the column. This can be achieved using a linear gradient of aqueous solution of sodium chloride.

Pure form of bFGF can be obtained by the method of the present invention. In particular, can be obtained 146-amino acid form of bFGF, designated as (10 155) bFGF. Received bFGF can be used for the treatment of wounds and burns, for example, bFGF can be used for wounds and burns in any padavitel.

The following examples illustrate the invention. Examples of obtaining and comparative examples.

Examples: getting 154/153 forms of bFGF

The construction sequence synthetic DNA for bFGF and expression plasmids carrying such sequencing was carried out according to the method described in EP-A-363375. The processes of fermentation and purification were carried out as follows:

(a) the fermentation Process

Bacterial strain, E. coli B, from the collection of the Pasteur Institute, was transformed with a plasmid carrying both the human gene encoding bFGF, and the gene for tetracycline resistance. This transformed strain was used to produce recombinant replicationmanager h-bFGF (human bFGF). Master Cell Bank cell (15 freeze-dried bubbles) and Working Cell Bank cell (W. B. C.) (70 bubbles stored in liquid nitrogen at 190oC) of this strain were obtained. The contents of one vial W. C. B. was used as inoculum for the fermentation.

The enzymatic process was carried out in 10 l fermentor filled with 4 liters of a nutrient medium. In the nutrient medium was added the hydrochloride tetracycline in order to maintain conditions VSL 2500 500 mg/l, as shown by measurement using a comparative gel electrophoresis.

During the enzymatic phase took the enrichment of pure oxygen in order to provide a large bacterial growth.

The original clean

Cells (microorganisms) were separated from the total enzyme mixture by centrifugation. The obtained pellets were re-suspended in sodium phosphate buffer containing sodium chloride. You had at least three passes through the homogenizer high pressure for effective cell breakage. The obtained cell lysate was clarified by centrifugation and the supernatant was collected for further processing.

Clean

The clarified supernatant was passed through a column of Sepharose (Trade Mark) S Fast Flow (cation-exchanger) and the product was blueraven from this column using a gradient of increasing concentrations of sodium chloride in phosphate buffer. Then the product was purified on a column of Heparin Sepharose 6B by elution with a gradient of increasing concentration of sodium chloride in phosphate buffer. Finally, was buffer exchanged on a Sephadex (Trade Mark) G-25 resin to obtain a product in the amount of food buffer (sodium phosphate ED sodium hydroxide. Heparin Sepharose was washed successively with solutions containing 3M sodium chloride at pH 8.5 and pH 5.5.

Thus was obtained 154/153 form of bFGF.

It is a mixture of approximately 50:50 from:

154-amino acid human bFGF /2 155/ bFGF having the amino acid sequence 155-amino acid forms, which describes Abraham et al and shown in SEQ ID NO, but without the N-terminal Met residue; and

153-amino acid human bFGF /3 155/ bFGF, consisting of a 155-amino acid forms of bFGF shown in SEQ ID NO: 1, but without the N-terminal Met residue.

Example 1. Heparansulfate as a protective agent; pepsin A.

1. Obtaining a protein sample

154/153 form bFGF preparative sample was obtained at a concentration of 1.8 mg/ml in a buffered solution:

10 mm monolatry phosphate

0.1 mm EDTA disodium salt,

pH 6,0.

In order to avoid any oxidation of the protein in a process controlled hydrolysis, was added 20 mg of dithiothreitol to 1.8 mg bFGF.

Methods of enzymatic hydrolysis of bFGF:

a) Standard solution of the protective agent

Solution 1: 20 mg protective solution in 1 ml of H2O

b) Preparation of solution of bFGF and protective AG is 2O,

18 mg (18 ml) pepsin A mucous membrane of pig intestines,

final volume: 200 µl.

Protective agent represented heparan sulfate and the ratio of bFGF / protective agent was 1:1 (weight/weight). Pepsin A (EC 3.4.23.1) was added to a solution of bFGF and protective agent at pH 4.0 and incubated with 10oC for 1 h

3. Kinetics of enzymatic hydrolysis

The procedure of hydrolysis was observed at different times using SDS-PAGE analysis. The samples of the mixture were analyzed after 1, 20, 40 and 60 min using SDS-PAGE. Department 154/153 bFGF forms from 146-bFGF forms was achieved in SDS-PAGE analysis using PHAST System (Trade Mark; Pharmacia). With regard to the sample buffer, the conditions of preparation of the sample were defined as follows in order to avoid any precipitation of protein from the sample in the filling area Phast Cell Higri Density:

15 μl of protein solution;

10 μl of 40 mm Tris/HCl, 4 mm EDTA, 10% SDS, 20% mercaptoethanol, pH 8.0;

3 μl of DMSO (dimethyl sulfoxide);

1 μl of Bromphenol blue (0,3%).

Denaturation of the samples was achieved by heating the sample at 100oC for 5 minutes Analysis of samples showed that samples taken from the treated mixture after GF recombinant fragment, used as a reference.

4. Purification of bFGF

The reaction mixture was directly filtered through a Heparin-agarose column pre-equilibrated Tris buffer 10 mm, pH 8.0, and 0.5 NaCl. bFGF was elyuirovaniya at 1.5 M NaCl using a gradient from 0.5 M to 3 M NaCl. The concentration of protein was determined by the method Bradford's using standard 154/153 bFGF.

Elektropitanie 146-form bFGF

The procedure used in elektrosetevaya analysis was similar to the procedure described PIoug et al (Anal. Biochem, 181, 33 39, 1989). Used membrane was an Immobilon PVDF (MiIIipor).

After electrophoresis the gel was balanced in a transitional buffer /10 mm CAPS / 3-[cyclohexylamino]-1-propanesulfonic acid, the pH of 11.0, 20% methanol) for 10 minutes Immobilon PVDF was initially moistened with methanol and then equilibrated in a transitional buffer before use. Was used semi electroepitaxy analysis (Kyhsel An dersen, J. Biochem. Biophys. Melhoass, 10. 203 209, 1984). Elektroprenos was carried out for 2 h at 0.2 mA/cm2.

PVDF membrane was washed in water and then dyed in 0.1% (wt/vol) Coomassie brilliant blue R 250 in 50% (vol/vol) methanol for 1 min to Excess) methanol, containing 10% (vol/vol) acetic acid for 10 minutes, the PVDF Membrane was then carefully washed in water before sequencing.

6. Analysis of the amino-terminal sequence

Clipping region PVDF membrane containing the colored protein, were placed as a single layer on top of Polybrene standard filter. Sequencing was carried out on the Determinant sequence Applied Biosystems Model 470A, supplied sequentially PTH-amino acid analyzer Model 120 A.

The first three amino acids NH2-terminal part of the protein defined under these conditions were:

Pro-Ala-Leu

These amino acids correspond to the three amino acids aminoterminal 146-bFGF forms.

7. Laser scanning densitometric analysis

This analysis was conducted to determine the percentage of 146-bFGF obtained after 1 h of hydrolysis 154/153-bFGF using pepsin A. After 1 h, 50 μl of the reaction mixture were mixed with 50 µl of SDS-denaturing buffer. Under the same conditions bFGF samples of different concentrations of protein were SDS-are denatured. These samples were separated by gel electrophoresis PAA 4/30 (Pharmacia). Was analyzed by densitometry city conditions, it was found that the concentration of 146-bFGF forms corresponded to 91% of the initial 154/153-bFGF concentration.

Exit 146-bFGF after affinity purification

The yield of the reaction enzymatic hydrolysis was also determined after purification 146-bFGF forms on a heparin-agarose column of 1.64 mg of the reaction mixture from p. 2. above, was passed through a heparin-agarose column pre-equilibrated 10 mm Tris, pH 8, 0.5 M NaCl. At pH 8 pepsin A totally inactive, and the complex between heparan sulfate and bFGF destabilized, allowing it to be adsorbed bFGF to heparin-agarose present in excess in the environment. bFGF was blueraven at 1:5 M NaCl. Was allocated to 1.14 mg 146-bFGF forms. The total output 146-bFGF amino acid form after controlled hydrolysis using heparan sulfate as a protective agent and purification on affinity column was 73%

Example 2. Heparin as a protective agent; pepsin A.

Was repeated procedure of example 1, except that the protective agent was heparin, the ratio of bFGF-heparin was 5:1 (weight/weight), aliquots of the processed mixtures were analyzed after 2 min and 4, 6 and 8 h and the total incubation time was 8 hours a Solution of bFGF and heparin consisted of:

100 μl protein A,

final volume: 200 µl.

Solution 2 consisted of 100 μl of solution 1 and 900 μl of H2O. Under these conditions, only one fragment was obtained after 8 h of incubation. This fragment had apparent molecular weight 16.200, as determined by SDS-PAGE analysis.

Using analysis aminoterminal sequences were obtained for three of the same N-terminal amino acid, as in example 1: Pro-Ala-Leu.

Example 3. Heparin as a protective agent; pepsin A.

The output of the controlled hydrolysis of bFGF, anti-heparin was also determined after purification 146-bFGF fragment on a heparin-agarose column. After 7 h of hydrolysis in the same conditions as in example 2, the reaction medium containing 1,54 mg bFGF was filtered through a heparin-agarose column pre-equilibrated with 10 mm sodium phosphate buffer, pH 8, and 0.5 M NaCl. 1,02 mg 146-bFGF was allerban with 1.5 M NaCl.

The total output 146-bFGF amino acid form after purification by affinity column using heparin as a protective agent was 69%

Example 4. Heparin immobilized on Agarose as a protective agent; pepsin A.

1. The procedure enzymatic hydrolysis bFGF

Heparin-agarose co and 10oC. 154/153-bFGF was passed through the affinity column. Then 0.18 mg of pepsin, diluted with the same phosphate solution was passed through the column. The enzyme was recirculates through the column at a flow rate of 0.5 ml/min for 6 hours

When time is 6 h, the column was washed with 10 mm sodium phosphate buffer pH 7, 0.1 mm EDTA, 0.5 M NaCl, (3 volumes), then gradient from 0.5 M to 3 M NaCl. bFGF was blueraven with 1.5 M NaCl.

2. Analysis aminoterminal sequence

After electrophoresis the gel was exposed to electropolishing on Immob: 10n PVDF using a semi-absorbing device as described in example 1. Clipping region PVDF membrane containing the colored protein, were placed in a single layer on top of Polybren standard filter. The first three amino acids NH2-terminal part of the protein defined under these conditions, were: Pro-Ala-Leu.

These amino acids correspond to the three amino acids from aminoterminal part 146-bFGF forms.

3. Exit when allocating 146 bFGF

The yield of the reaction of hydrolysis was determined after quantitative determination 146 bFGF fragment, washed from the heparin-agarose column. 1.28 mg 146 - bFGF was selected. Total yield "Obtention" 81%

Example 5. Heparin as a protective agent; cathepsin D.

1. The procedure enzymatic hydrolysis bFGF

Was repeated procedure of example 2, except that instead of pepsin was used cathepsin D from the liver of the ox, and the incubation time was 110 hours

2. Reaction kinetics

Contents 146 bFGF fragment obtained during enzymatic hydrolysis, was very low, but detektivami by SDS PAGE analysis after 1 h After 72 h hydrolysis 154/153 bFGF was not full and 36 µg cathepsin D was added to the reaction medium. After 40 h, the reaction was completed. This is due to low activity of cathepsin D (10 units/mg).

6. Analysis aminoterminal sequence

After electrophoresis the gel was exposed to electropolishing on Immobilon PVDF using "Siemi-dry blotting assembly, as described in example 1. Clipping region PVDF membrane containing the colored protein, were placed in a single layer on top of the Poly bren standard filter. Sequencing was performed on an Applied Biosystems analyzer determinant sequence Model 470A, connected in series with the PTH-amino acid analyzer Model 120A. The first three amino acids NH2terminal the protective agent.

Example 1 was repeated except that was not present protective agent. 154/153 form of bFGF has been completely redesigned pepsin within A few minutes.

Comparative example 2: cathepsin D without protective agent.

Example 5 was repeated except that the protective agent was not present. 154/153 form of bFGF was completely redesigned by cathepsin D within a few minutes.

Comparative example 3: -chymotrypsin.

Example 1 was repeated, except that instead of pepsin And used a-chymotrypsin, and the protective agent was not present. 154/153 form of bFGF was completely redesigned a-chymotrypsin within a few minutes. Were therefore tested various anionic compounds with respect to their ability to protect bFGF from a-chymotrypsin:

1. Heparin 3.000 (Sigma, H 7516)

the ratio of bFGF / protective agent 1,

temperature 10oC,

pH 7.5,

the incubation time of 4 hours.

When using heparin 3.000 as a protective agent was obtained mainly one piece of the apparent molecular weight of 14,000 and a few other fragments of lower molecular weight as determined by SDS-PAGE analysis.

2. Gurama incubation for 4 h

When using this heparin properties as a protective agent protection was not appropriate, and there were many fragments.

3. Chondroitin sulphate

the ratio of bFGF / protective agent (weight/weight) 1,

temperature 10oC,

pH 7.5,

incubation time 2 h

Protection was not suitable, and there were many fragments.

4. Dermatan sulfate

the ratio of bFGF / protective agent (weight/weight) 1,

temperature 10oC,

pH 7.5,

incubation time 2 h

Protection was not appropriate, and there were many fragments.

5. Poliasparaginovaya acid

the ratio of bFGF / protective agent (weight/weight) 1,

temperature 10oC,

pH 7.5,

incubation time 2 h

When using poliasparaginovaya acid as a protective agent protection was not appropriate, and there were many fragments.

Example 6: Heparin or heparan sulfate as a protective agent; pepsin And or cathepsin D

1. Experimental Protocol

Proteolytic processing of soluble complexes of heparin and bFGF bFGF - heparin sulfate solution

1.6 mg 154/153-amino acid forms of bFGF were notorious with 1.6 mg of heparin or heparan-Ino 160 μg of pepsin A (Sigma, P-6887, 3200 4500 units/mg protein). At various times samples were taken of the reaction medium and directly passed through a heparin - separato column, pre-equilibrated with 10 mm phosphate buffer pH 8.0/0.5 M NaCl at 4oC. the Column was then intensively washed with the same buffer, and bFGF was blueraven 3 M NaCl in 10 mm phosphate buffer pH 8.0. The collected fractions were desalted on a column of Sephadex C 25 pre-equilibrated with 10 mm phosphate buffer pH 6.0 and analyzed SDS PAGE analysis. When using heparan sulfate as a protective agent was obtained 146 bFGF with quantitative yield after 1 h Treatment with pepsin complex of heparin-bFGF led to the same quantitative yield after 6 h incubation time.

Treatment with pepsin A bFGF associated with heparin-sepharose column

50 mg bFGF / 154/153 have been passed through a heparin-separato column (Pharmacia) (2.6 x 22.5 cm), previously equilibrated to 25 mm citrate buffer pH of 4.0/0.5 M NaCl at 4oC and at a flow rate of 2.5 ml/min. a Solution of 680 units/ml porcine pepsin A (Sigma) in citrate-phosphate buffer continuously recycled through the column at 2.5 ml/min for 3 h at 4oC. the Column was then intensively washed with 25 mm phosphatase pH 8.0. bFGF-containing fractions were collected, concentrated by ultrafiltration on Amicon PM 10 membrane and desalted on a Sephadex C-25 column (Pharmacia), previously equilibrated with 10 mm phosphate buffer pH 6,0.

Analysis of the N-terminal sequence

Automated analysis of N-terminal sequencing was carried out on a Model 477A Pulsedliquid Phase Sequence (Applied Biosystems, CA, USA) with a serial Model 120 A PTH analyzer. We used Normal-1 with minor modifications. All materials to determine the sequence and reagents were purchased from Applied Biosystems.

Analysis of the C-terminal sequence

Investigation of flow effects carboxypeptidase on bFGF in time was carried out at room temperature in 10 mm sodium acetate buffer pH 3,8/a 0.05% Brij 35, using the ratio of enzyme / substrate of about 1: 100 (weight/weight) (Lu et al, J. Chromatogr. 447, 351 364, 1988).

Enzymatic processing 0,5 1 nmol of protein was carried out for 2 h with 0.1 to 0.2 µg CpP (Boehringer); N-Leucine was added as an internal standard. At various times were selected aliquots 10/100 µl and subjected to amino acid analysis after automated PITC derivatization on derivatograph Model 420 A (Applied Biosy is yserowanej PTC-amino acids on PTC-C8 column (P/N 0711-0204, 22 x 2.1 mm, 5 µm, Applied Biosystems).

Bioassays

To study the proliferative response induced by bFGF was used endothelial cell strain obtained from the aorta of a bull (BAEC). Cells were inoculated at 2500 cells / cell in 96 cellular microtitration plate, in a nutrient medium consisting of Dulblecco''s Modified Eagle's Medium (MEME), supplemented with 13% of fetal serum of bovine (FBS) (G: bro, UK). After attaching the nutrient medium was replaced with experimental medium consisting of DMEM supplemented with 0.5% fetal calf serum (FBS), 0.1% of bovine serum albumin (BSA) (Sigma, USA), and the desired concentrations of bFGf (Erbamont). Cultures were incubated for 3 days, and at that time they were mixed with formalin and stained with 0.5 crystal violet. After staining, the cells were thoroughly washed to remove those that are not dye. Methanol (95% 0.1 ml/cell) was added to each cell for the extraction of the dye is proportional to the number of cells grown on cell. Plates were transferred automatically read in spectrophotometric macroplastique reader, equipped with a 540 nm filter.

For the synthesis of plasminogen asset the environment, which was replaced after insertion in DMEM supplemented with 0.5% (FBF), 0.1% of BSA and the test concentrations of bFGF. After incubation for 28 h of culture were washed and cells were visualized with a solution containing 0.5% Triton X-100. Aliquots of cell lysates were analyzed for plasminogen activator activity using a chromogenic substrate (Spectrosyme PL) and plasminogen (both reagents from Ammerican Diagnostica Inc) for amiloliticescoe analysis.

2. Results

Controlled enzymatic treatment bFGF

Purified recombinant 154/153 the mixture was incubated with two different the aspartic proteases: pepsin a and cathepsin D. Aliquots of the reaction mixture were taken at various time intervals and subjected to SDS-Page analysis showing that in the absence of any protective agent bFGF was rapidly cleaved into small peptides. In contrast, treatment of bFGF / 154/153 / pepsin A 10:1 pH 4,0; 10oC/ in the presence of heparin or heparan-sulfate (1:1 weight/weight) resulted in progressive and full conversion 154/153-amino acid forms in the form of lower molecular weight, which migrates together with our standard 146/145 amino acid form.

After Electromechanica automated analysis of N-terminal sequence on "pulsed" liquid-phase determinant sequence. The first three cycles resulted in a single homogeneous sequence; Pro-Ala-Leu, which corresponds to the intact N-terminal end of the famous 146-amino acid forms. No other sequence has not been determined, showing that despite the presence of three Leu-Pro sites on the molecule bFGF, when the elongated forms of bFGF were comprehensively associated with heparin or heparan-sulfate, enzymatic treatment with pepsin A split specifically and only the peptide bond Leu9Pro10.

Controlled proteolytic cleavage NH2-extended "protected heparin molecules bFGF, as obtained by pepsin A, was achieved after treatment with cathepsin D, although it took a longer incubation time for this proteolytic reaction, possibly because of the lower specific activity of the enzyme used. When under similar conditions, but at a pH of 7.5 was added chemotrypsin was detected after gelelectrophoresis incubated mixture of a single polypeptide of approximately 14000 Yes, and there was no evidence of the presence of the 146-amino acid forms.

Enzymatic processing of bFGF on a wide scale in the heparin-sepharose column

Rezultati 154/153 mixture elongated bFGF, associated with heparin-sepharose column. Accordingly, 50 mg bFGF (154/153) were passed through a column of heparin-sepharose, pre-balanced citrate-phosphate buffer pH of 4.0. A solution of pepsin A recycled continuously through the column for 3 h at 4oC. the column was washed with phosphate buffer at alkaline pH as for the inactivation and removal of the enzyme. The obtained polypeptide was elyuirovaniya from column 3 M NaCl in phosphate buffer at pH 8.0. The fractions containing bFGF were collected and desalted on a column of Sephadex G-25.

The collected fractions were analyzed on SDS PAGE showed a single band with molecular weight corresponding to a molecular weight standard 146/145 forms of bFGF. Analysis on HPLC with a reverse phase also resulted in a single peak. Analysis of the N-terminal sequence resulted in a single sequence corresponding to the correct, intact N-terminal end of the molecule bFGF, i.e. Pro-Ala-Leu-. Analysis of the C-terminal sequence showed the expected end of the bFGF molecule, i.e.- Ala-Lys-Ser. Thus, also, when bFGF was associated with heparin-sepharose resin, pepsin A was able to specifically cleave the molecule at Leu9- Pro10communication, generating th is 54/153 amino acid form was compared with the activity of homogeneous 146-amino acid form, obtained using the described proteolytic process. Two activity, induction of proliferative response and synthesis plasminogen activator were studied in endothelial cells of the aorta bull (BAEC). Both tests confirmed the in vitro biological equivalence 154/153 compared with 146 amino acid form, obtained through enzymatic process.

THE LIST OF SEQUENCES

// INFORMATION FOR SEQ ID NO:1:

// SEQUENCE CHARACTERISTICS:

/A/ LENGTH: 155 amino acids

/B/: TYPE: amino acid

/C/ "Strandedness" single

/D/ TOPOLOGY: linear

/II/ TYPE MOLECULES: protein

// SEQUENCE DESCRIPTION: SEQ ID NO:1:

/2/ INFORMATION FOR SEQUENCE SEQ ID NO:2:

// SEQUENCE CHARACTERISTICS:

/A/ LENGTH: 8 amino acids

/B/: TYPE: amino acid

/C/ "Strandedness" single

/D/ TOPOLOGY: linear

/II/ TYPE of MOLECULE: peptide

// SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met-Ala-Ala - Gly-Ser-Ile-Jhr-Jhr

/3/ INFORMATION FOR SEQ ID NO:3:

I) SEQUENCE CHARACTERISTICS:

/A/ LENGTH: 7 amino acids

/B/: TYPE: amino acid

/C/ "Strandedness" single

/D/ TOPOLOGY: linear

// TYPE of MOLECULE: peptide

// DESCRIPTION IS EGOVERNANCE:

/A/ LENGTH: 6 amino acids

/B/: TYPE: amino acid

/C/ "Strandedness" single

/D/ TOPOLOGY: linear

// TYPE of MOLECULE: peptide

// SEQUENCE DESCRIPTION: SEQ ID NO:4:

< / BR>
/6/ INFORMATION FOR SEQ ID NO:5

// SEQUENCE CHARACTERISTICS:

/A/ LENGTH: 5 amino acids

/B/: TYPE: amino acid

/C/ "Strandedness": single

/D/ TOPOLOGY: linear

// TYPE of MOLECULE: peptide

// SEQUENCE DESCRIPTION: SEQ ID NO:3:

< / BR>
/6/ INFORMATION FOR SEQ ID NO:6:

// SEQUENCE CHARACTERISTICS:

/A/ LENGTH: 4 amino acids

/B/ TYPE: amino acid

/C/ "Standedness": single

/D/ TOPOLOGY: linear

// TYPE of MOLECULE: peptide

// SEQUENCE DESCRIPTION: SEQ ID NO:6:

n

1. The enzymatic method of obtaining basic fibroblast growth factor bFGF (10 155), including the interaction of bFGF with a protective agent with a ratio of protective agent bFGF from 0.5 to 1 1 to 10 (weight/weight), the formation of the complex between the protective agent and bFGF, the processing of the received complex proteolytic enzyme, characterized in that as bFGF use bFGF (2 155) and/or bFGF (3 155), as a protective agent heparin or heparan-sulfate, and the resulting complex process PE fact, that bFGF (2 155) and/or bFGF (3 - 155) is a human.

3. The method according to p. 1 or 2, characterized in that the protective agent and bFGF (2 - 155) and/or bFGF (3 155) is added to the buffer solution.

4. The method according to PP.1 to 3, characterized in that the heparin or heparan-sulfate immobilized on the substrate with the formation of affinity columns, column loaded with a solution of bFGF (2 155) and/or bFGF (3 155), then passed through the column a solution of pepsin or cathepsin D, derived bFGF (10 155) elute.

5. A pharmaceutical composition comprising a major factor in the growth of fibroblast (bFGF) and a pharmaceutically acceptable carrier or diluent, characterized in that as bFGF use bFGF (10 155), obtained by the interaction of bFGF (2 155) and/or bFGF (3 155) with a protective agent, heparin or heparan-sulfate, with a ratio of protective agent bFGF from 0.5 to 1 1 to 10 (weight/weight), the complex formation between the protective agent and bFGF, processing the received complex pepsin And or cathepsin D and the allocation of bFGF (10 155).

 

Same patents:

The invention relates to molecular biology, in particular the production of insulin from predecessor
The invention relates to medical Microbiology, and in particular to methods of obtaining protein hydrolysates, and can be used to obtain the basic nutrient medium for the accumulation and cultivation of microorganisms bacterial nature

The invention relates to the field of protein extraction from natural sources

The invention relates to the field of biotechnology and relates to a method of obtaining ovomucoid (Ω) of the whole protein eggs (CBA)

The invention relates to the field of biotechnology and relates to a method of obtaining ovomucoid (Ω) of the whole protein eggs (CBA)

The invention relates to organic chemistry, in particular to preparative peptide synthesis of GnRH analogues in solution

The invention relates to the processing of vegetable raw materials, namely the method of obtaining protein hydrolysates, for example, from soybean meal and algal waste agar production, which can be used for food or as supplements in animal feed

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

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: peptides, pharmacy.

SUBSTANCE: invention relates to a new method for preparing a pharmaceutical composition for the parenteral administration in mammals that comprises salt of difficulty soluble peptides. Method involves treatment of the parent readily soluble acid-additive salt of the basic peptide of LHRH antagonist in the presence of suitable diluting agent with the mixed ion-exchange resin or mixture of acid and basic ion-exchange resins to form free basic peptide. Then ion-exchange resin is removed and free basic peptide is treated with inorganic or organic acid to form the final product, required acid-additive peptide salt followed by addition of suitable pharmaceutical vehicles and/or filling agents and removal of a diluting agent.

EFFECT: improved preparing method.

5 cl, 1 dwg, 1 ex

FIELD: feed mill industry.

SUBSTANCE: method involves treating sunflower oilcake with catholyte; removing treated solution; extracting protein; filtering; drying sediment; grinding; simultaneously with treatment of sunflower oilcake with catholyte, providing treatment of soya with anolyte, with catholyte and anolyte circulating at equal velocities. Apparatus has two chambers connected with each other through semi-permeable partition. Each of said chambers is equipped with electrodes and dc source. Apparatus is further equipped with collecting chamber and pumps with pipelines.

EFFECT: increased efficiency of method and apparatus, reduced production time and decreased costs of additives.

1 dwg, 1 tbl

FIELD: biotechnology.

SUBSTANCE: method involves purification of protein raw, addition of water and ice, milling a mixture followed by hydrolysis. After hydrolysis collagen-containing solution is homogenized and collagen is separated. Hydrolysis is carried out for two stages. The first stage is carried out by treatment of the reaction mixture with lipase from fungus Rhizopus oryzae, and at the second stage a proteolytic enzyme as neutral protease is used. Invention provides preparing collagen approaching to natural collagen by physicochemical and structural-mechanical properties. Invention can be used in food processing industry, cosmetic, medicinal and other branched of industry.

EFFECT: improved preparing method.

4 cl, 3 ex

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

Up!