The method of obtaining the primary proteinase inhibitor from the bodies of cattle

 

(57) Abstract:

The invention relates to biotechnology, in particular to a technology for basic proteinase inhibitor from the bodies of cattle. The technical result from the application of the invention consists in the selection of the main proteinase inhibitor for medical purposes from a wide range of solutions: extracts of organs, the crude solutions containing high concentrations of salts. The proposed method of receiving basic proteinase inhibitor is that the crude solution obtained from the bodies of cattle grinding, extraction, separation of the cake, removing proteins and lipids, and the lead adsorption on macroporous polyacrylic the cation-exchanger with carboxyl groups at pH 7-10,5 from solutions with a conductivity of not more than 6 mS followed by desorption of the target product salt solution at pH 1-13 and his selection of well-known techniques. table 1.

The invention relates to biotechnology, in particular to a technology for basic proteinase inhibitor (type Konitza) from the bodies of cattle, and can be used in the medical industry for the preparation of medicines, ispim technical solution is the way to obtain basic proteinase inhibitor, namely, that his adsorb from solution of the amphoteric ion exchanger based on polystyrene containing aminouksusnoy or iminoxyl group, with the subsequent removal neabsorbirowawrzayasa substances by flushing the buffer or salt solution and desorption of the inhibitor by creating a salt gradient.

The disadvantage of this method is the use of pre-treated (2000-4000 KIE/mg protein) and highly concentrated solutions (5100-7800 KIE/ml) of the main proteinase inhibitor with a very low salt content (conductivity of 0.12 mS), that is, their pre-concentration and desalting. The specific activity of the product increases 1,23-3.1 times.

The expected technical result from the proposed method is that the selection of the main proteinase inhibitor can be from a wider range of solutions: from extracts obtained directly from the bodies of cattle, the poor content of the inhibitor and with its low specific activity; from treated and untreated solutions with conductivity up to 6 mS; from solutions containing organic solvents.

The proposed method for extracting the CSOs cattle grinding, extraction, separation of the cake, removing proteins and lipids, the adsorption of the inhibitor is carried out at pH 7.0 to 10.5 from solutions with a conductivity of 6 mS on the cation-exchanger with carboxyl groups and desorbed target product salt solution at pH 1-13 and select the known techniques.

As carboxylic kationoobmennikom most suitable modern macroporous polyacrylic, which are stable in a wide pH range, have good hydrodynamic properties in acidic and alkaline conditions, have high selectivity and capacity in relation to the main inhibitor of proteinases.

Salient features of the invention are the use of the crude solution, which is obtained from the bodies of cattle grinding, extraction, separation of the cake, removing proteins and lipids, the adsorption of the inhibitor are on macroporous polyacrylic Latinoamerica with carboxyl groups at pH 7-10,5 from solutions with a conductivity of not more than 6 mS followed by desorption of the target product salt solution at pH 1-13 and his selection of well-known techniques.

The specific activity of the primary inhibitor of proteinases secreted by the proposed cattle are ground in a meat grinder and the extraction is carried out within 2 h 3 l 65% aqueous solution of ethanol at pH 4.0, which is supported by means of phosphoric acid. After separation of pomace extract is subjected to vacuum distillation until the ethanol content of 20% lipid Layer formed upon cooling to 5oC, is separated by filtration. Using calcium hydroxide pH 7.0 set and after 2 h the solution is filtered. The resulting solution having a conductivity of 2mS and content of inhibitor 321 KIE/ml (29 KIE/mg protein), passed through a cation exchange resin Bio-Rex 70 (Bio-Rad, USA) placed in column 1,5x8,0 see the Target product elute solution of sodium chloride with a pH of 7.0. The fractions containing the active protein are pooled. The specific activity of the main proteinase inhibitor during chromatographic purification on carboxylic cation exchanger increases 70 times and is 2030 KIE/mg protein.

To the combined solution was added sodium chloride to a concentration of 200 g/l and stirred until the salt is completely dissolved. After 12 h, the precipitate was separated by centrifugation and dissolved in 15 ml of hydrochloric acid with a pH of 1.7, applied on a column with Sephadex G-50f (1,h cm) and lead chromatography in a solution of hydrochloric acid with a pH of 1.7. Fractions of the main proteinase inhibitor, purified from high molecular weight proteins are pooled and subjected to a sterilizing filtrowanie and dried at 20oC to constant weight. Receive 12 mg pyrogen-free powder main proteinase inhibitor with activity 5000 CUE/mg, is suitable for the preparation of dosage forms.

Example 2. 1 kg frozen lungs are crushed and extracted with 2 5 l of a solution of phosphoric acid to pH 4.0. Separate the lung tissue by centrifugation and the extract is heated to 70oC, and then cooled. After adjusting the pH to a value of 5.6 solution of 20% sodium hydroxide to give the solution to precipitate, and separating the precipitated precipitate by filtration. In the supernatant using sodium hydroxide solution set pH of 9.5, while the conductivity of the solution was 6 mS, and the specific activity of 33 KIE/mg of the resulting solution is passed through a carboxylic cation exchanger Bicarb-t (Biochemical, Latvia), placed in column 1,5x8,0 see the Elution of the main proteinase inhibitor spend 1 M solution of sodium chloride with a pH of 12.5. The specific activity of combined fractions containing the inhibitor, when chromatographic purification increases to 75 times compared to the initial solution and is 2475 KIE/mg protein. To the combined solution was added sodium chloride to a concentration of 250 g/l mix until the salt is completely dissolved. After 12 h dropped osadia ammonium carbonic acid. Spend chromatography on Sephadex G-50f (2,h cm) in a solution of 0.05 M ammonium carbonic acid. At the exit of the column fractions containing inhibitor, combined and subjected to freeze drying. Get 100 mg white pyrogen-free powder main proteinase inhibitor with activity 5100 KIE/mg, is suitable for the preparation of dosage forms.

Examples 3-9. 1 kg of the bodies of cattle, containing the main inhibitor of proteases, ground in a meat grinder, extracted with 5 l of a solution, supporting with sulfuric acid pH 3.0. Meal bodies separated by filtration. Using a 20% aqueous solution of sodium hydroxide pH 7.0 set and loose precipitate was separated by centrifugation. To clear the extract was added sodium chloride to a concentration of 250 g/l After dilution of the salt solution stand for 12 h and precipitated precipitate was separated by centrifugation. Protein is dissolved in 100-500 ml of distilled water to achieve a conductivity of 3 mS and establish a pH of 7-10,5. To the resulting solution was added 15 ml of cation exchanger Amberlit IRC-50 (R H, USA), equilibrated with the same pH value. The solution is stirred for 1 h and separated media filtration. The elution of lead 1 M solution of sodium chloride with a pH of 9-11,5 and Obyedinennaya to its concentration of 550 g/l and stirred until the salt is completely dissolved. After 10 h, the precipitate was separated by centrifugation and dissolved in 10 ml of a 5 aqueous solution of acetic acid. Spend chromatography on hydrophilic gel ED-5,0 (1,h cm) in a solution of 5% acetic acid. Faction inhibitor unite, is subjected to sterilizing filtration and lyophilized. Get pyrogen-free white powder main proteinase inhibitor.

The results of the chromatographic purification and characterization of the final product are summarized in the table.

The method of obtaining the primary proteinase inhibitor adsorption from solutions with subsequent desorption, characterized in that the use of untreated solutions of the main proteinase inhibitor, which is obtained from the bodies of cattle by crushing raw materials, extraction, separation meal, removal of proteins and lipids, and the lead adsorption on macroporous polyacrylic Latinoamerica with carboxyl groups at pH 7 to 10.5 from solutions with a conductivity of not more than 6 mS followed by desorption of the target product salt solution at a pH of 1 to 13 and its selection of well-known techniques.

 

Same patents:

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

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

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

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