Method of production of cytochrome c

FIELD: biotechnology.

SUBSTANCE: method comprises degreasing of hearts of pigs, cattle or horses. The prepared raw material is ground to obtain minced meat, then extracted with 2.5% trichloroacetic acid solution at the temperature of 10-15°C and pH of 3.9-4.2. The extract is separated and its sorption is carried out on the cation exchanger Nuvia™ S, equilibrated with distilled water. After completion of sorption the cation exchanger Nuvia™ S is washed with distilled water and ammonia-ammonium buffer solution. Then cytochrome C is eluted with from the cation exchanger Nuvia™ S with ammonium sulphate solution followed by its purification by diafiltration until complete removal of sulphate ammonium ions and concentration of cytochrome C by ultrafiltration.

EFFECT: invention enables to increase the yield of product, increase its purity and reduce the technological process.

3 cl, 1 tbl, 3 ex

 

The present invention relates to biotechnology, the field of production of medicinal protein preparations, namely, the method of production of cytochrome C from heart muscles mammals - pigs, cattle and horses.

Cytochrome C is an enzyme with a molecular weight of about 12700 Yes, as a prosthetic group contains hematomas iron catalyzes oxidation-reduction reactions and serves as a carrier of electrons (Y.A. Ovchinnikov. Bioorganic chemistry. M.: Chemistry, 1987, s).

Cytochrome C is widely used in medicine and veterinary medicine as a medicinal product, and as a reagent in biochemistry and biotechnology.

The invention may be used upon receipt of cytochrome C on an industrial scale.

Cytochrome C can be selected from various kinds of natural raw materials: yeast, rice, heart muscles mammals (horses, cattle, pigs, dogs, rabbits), birds (pigeons, penguins, hearts, salmon and others. Known methods for producing cytochrome C, including the allocation of the heart muscle mammal, preferably cattle, by solvent extraction and sorption purification of the extract by using a magnesium aluminosilicate (F. Okumura and al. Chem. Soc. Japan. V.49, No.1, p.65, 1975) or carboxylic cation exchangers of the type Amberlite HEH - 64 (B. Hagihara and al. J. Biochem V. 45, No. 8, p.551, 1975) and CMDM (L.K. Shataeva, H.H. Kuznetsova, GA the El'kin Yury Evgen'evich. Carboxylic cation exchangers in biology. Leningrad: Nauka, 1979, s.285).

These sorbents are characterized by small size and complexity of industrial applications, especially carboxylic cation exchange resin due to the difference in the degree of swelling in the H+and Na+form.

Closest to the proposed invention is a method for cytochrome C from heart muscles mammals - pigs, cattle and horses (Patent RU 2435597, publ. 10.12.2011, bull. No. 34), including degreasing hearts of pigs, cattle or horses, the removal of ligaments and blood vessels, grinding and extraction of a 2.5% solution of trichloroacetic acid at a temperature of 10-15°C, at a pH of 3.9 to 4.2, and the separation of the extract from the stuffing. Sorption of the target product are conducted directly from the filtered extract at a pH of extract on the cation exchanger UNOsphere™ S balanced starting buffer solution containing 2 mm of citric acid and 1.5 mm Tris (Tris (hydroxymethyl) aminomethane) at pH 3.9 to 4.2, and after the sorption of the cation exchanger is washed with the starting buffer solution, ballast proteins are removed from the cation exchanger UNOsphere™ S 80 mm Tris solution and then spend the elution of the cytochrome C solution of ammonium sulfate with a concentration of 0.3 M, after the stage of elution carry out the deposition of ballast proteins by adding dry with whom lipata ammonium based 0,270 gml

In the prototype using a cation exchanger UNOsphere™ S (UNOsphere Polymer Technology, the production of "Bio-Rad USA) - having as functional groups - SO3group.

The disadvantage of this method of obtaining cytochrome C is a low degree of purity of cytochrome C, an indicator of purity (E550280) cytochrome C in the eluate (the ratio of the extinction of the solution of cytochrome C at a wavelength of 550 nm and a solution of cytochrome C at a wavelength of 280 nm) is 0.92-0,93 that requires deposition of ballast proteins from the eluate using the increased amount of ammonium sulfate (0.27 gml), also significant disadvantages of the above method of obtaining cytochrome C is to use solutions, which include expensive reagents, such as citric acid and Tris(hydroxymethyl) aminomethan. These shortcomings significantly reduce the efficiency and effectiveness of the process.

Thus, the present invention is to provide a method of producing cytochrome C from the extract using ion exchangers of the new generation, providing a high sorption capacity and reversibility of sorption with the use of inexpensive reagents, providing a high degree of purity of the target product, which makes them preferable for use in an industrial scale.

The technical is a mere result of the present invention is to increase the capacity of the target product, the increase in the purity of cytochrome C reduction process.

According to the present invention is proposed sorption method of producing cytochrome C from the extract using cation of a new generation Nuvia™ S (Nigh - Capacity Cation Exchange Media, the production of "Bio-Rad USA) containing as functional groups - sulfopropyl. During the final stages of obtaining cytochrome C used diafiltration to remove ions of ammonium sulfate) and ultrafiltration (for concentration of cytochrome C) in a tangential flow membranes with a limit exceptions molecular weight 5 kDa firm Millipore or Sartorius.

To achieve the technical result of the proposed method, which includes the following stages:

From the hearts of pigs, cattle or horses after degreasing and removal of ligaments, blood vessels, by grinding receive the stuffing. The resulting meat is extracted with a 2.5% solution of trichloroacetic acid at a temperature of 10-15°C, at a pH of 3.9 to 4.2. After separation from the meat directly lead sorption of the target product from the filtered extract by cation Nuvia™ S cation exchange resin pre-balance distilled water. After sorption, cation Nuvia™ S washed with distilled water. Removal of ballast proteins with columns hold of 0.075 M ammonium Ammon the emergency buffer solution pH of 10.5. The elution of cytochrome C from the cation exchanger Nuvia™ S conduct with a solution of ammonium sulfate with a concentration of 0.25 M For the Department of cytochrome C from ammonium sulfate are diafiltration in tangential flow membranes with a limit exceptions 5 kDa, the same ultrafiltration membranes provide a concentration of cytochrome C.

For equilibration and washing the column, the best option is distilled water. To remove the ballast protein from the cation exchanger is optimal 0,075 M ammonia / ammonium buffer solution pH=10,5, at lower concentrations and pH of the buffer solution is not completely washed out of the ballast proteins, higher concentrations and pH does not have a significant impact on the number of remote ballast proteins. For elution of cytochrome C from the cation exchanger is the most optimal solution of ammonium sulfate with a concentration of 0.25 M, a low concentration of ammonium sulfate is not completely elute the target product with the cation exchanger, at higher concentrations of the cation exchange resin partially suiryudan impurity proteins, which significantly reduces the degree of purity of the target product.

The use of cation Nuvia™ S, you can directly lead sorption of cytochrome C from the extract (at a pH of extract), use a smaller amount of the cation due to the greater its capacity to significantly increase the degree of purity citoh the OMA C. The increase in the purity of cytochrome C at a stage of desorption from the cation exchanger Nuvia™ S makes it possible exception stage of deposition of ballast proteins that significantly increases the effectiveness and efficiency of the process by reducing the consumption of reagents and by reducing the time of the process.

The present invention is illustrated by the following examples.

Example 1.

2.6 kg of meat derived from cardiac muscle of pigs after removal of blood vessels, ligaments and grinding, washed with water from the remnants of blood, extracted with 2.5% trichloroacetic acid. Extraction was carried out for 2 h at 10-15°C. the Extract is separated from the feedstock by filtration through a cotton-gauze filter with subsequent centrifugation at n=3000 rpm / min for 10 minutes the pH of the resulting extract to 4.1. Just get 2750 ml of the extract. The extract obtained is passed through a cation exchanger Nuvia™ S balanced distilled water. The column volume 49 ml (diameter 2.5 cm, height of the layer of sorbent 3.8 cm, the amount of sorbent 19 ml). Sorption is carried out at a throughput rate of solution 2240 ml/h Time of sorption of about 1.2 hours and Then the cation Nuvia™ S washed with 200 ml of distilled water. Washing time 0,09 hours For removal of ballast proteins from the column is passed 400 ml of 0.075 M ammonium ammonium buffer solution pH=10.5V at a speed of 750 ml / HR. Washing time of 0.53 hours

The elution is of akroma spend With a solution of ammonium sulfate with a concentration of 0.25 M. The feed rate of eluting solution of 75 ml/h elution 0.5 hours the Eluate selected fractions. Faction, red, unite. The volume fractions of about 25 ml. Indicator of the purity of cytochrome C (E550280) amounts to 1.15. Yield 245 mg of cytochrome C or 94.6 mgkg of raw material.

Example 2.

Is performed under the conditions of example 1. For the Department of cytochrome C from the ions of ammonium sulfate are diafiltration. For this purpose a solution of cytochrome C in the amount of 125 ml (combined fractions 5xdownload pass by ultrafiltration installation mode diafiltration in tangential flow across the membrane of the company Millipore (cassette type Pellicon XL) or Sartorius (cassette type Vivaflow 50) with a limit of exclusion molecular weight of 5 kDa. Collect 135 ml of a solution containing the target product.

The indicator of the purity of cytochrome C (E550280) - 1,24. The output 232 mg of cytochrome C or 89.2 mgkg of initial raw material (in terms of per download).

Example 3.

Is performed under the conditions of example 1 and 2. Cytochrome C after diafiltration concentrated by ultrafiltration. 135 ml of a solution of cytochrome C (combined fractions 5xdownload after diafiltration) is passed to the ultrafiltration installation in a tangential flow across the membrane of the company Millipore (cassette type Pellicon XL) or Sartorius (cassette type Vivaflow 50) with a limit of exclusion molecular weight of 5 to the and. Get 45 ml of a solution containing the target product.

The indicator of the purity of cytochrome C (E550280) - 1,26. Output 225,7 mg of cytochrome C or 86,8 mgkg of initial raw material (in terms of per download).

Examples 4-9 in stages made in the conditions of example 1, 2 and 3. All experimental data presented in the table.

The index And mean raw materials of the heart muscle pig in example 1, 2 and 3;

B - raw from the heart muscle of cattle; raw material of the cardiac muscles of the horse.

1. The method of producing cytochrome C, including degreasing hearts of pigs, cattle or horses, the removal of ligaments and blood vessels, grinding and extraction of a 2.5% solution of trichloroacetic acid at a temperature of 10-15°C, at a pH of 3.9 to 4.2, and the separation of the extract from beef, sorption and elution of the cation exchange resin, removing the ballast proteins, concentration and purification by diafiltration and ultrafiltration to remove ions of ammonium sulfate, characterized in that the sorption of the target product are conducted directly from the filtered extract at a pH of extract on the cation exchanger Nuvia™ S balanced distilled water, after sorption, cation washed with distilled water, ballast proteins are removed from the cation exchanger Nuvia™ S 0,075 M ammonia / ammonium buffer solution of pH 10.5 and then spend is linked to cytochrome C from the cation exchanger Nuvia™ S solution of ammonium sulfate with a concentration of 0.25 M

2. The method according to claim 1, characterized in that the purification of cytochrome C from the ions of ammonium sulfate spend diafiltrate in tangential flow through the membrane to limit the exclusion molecular weight of 5 kDa.

3. The method according to claim 1, characterized in that the concentration of cytochrome C was carried out by ultrafiltration in a tangential flow through the membrane to limit the exclusion molecular weight of 5 kDa.



 

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