RU2014335C1 - Method of bovine serum albumin purification - Google Patents

Abstract

FIELD: biochemistry, biotechnology. SUBSTANCE: bovine serum albumin (BSA) is isolated from blood plasma by precipitation with ethanol, by change of pH and ionic strength. Proteolysis of extrinsic proteins is carried out additionally with trypsin in bicarbonate buffer, pH 7.8. BSA is separated by gel-filtration on Sephadex G-75. EFFECT: improved method of purification. 1 dwg

Description

 The invention relates to biological chemistry and can be used to increase the degree of homogeneity of commercial preparations of bovine serum albumin (BSA).

 BSA is widely used in biochemical and immunological practice to suppress non-specific adsorption of antibodies, to stabilize antigens, as a marker protein, etc. The purity of the used albumin, determined by the degree of its homogeneity, is one of the factors affecting the quality of the research.

 Currently, the following main methods are used to isolate BSA from plasma: salting out with inorganic salts (usually ammonium sulfate), precipitation by organic solvents at low temperatures, selective denaturation followed by precipitation of foreign proteins. Further purification of albumin in order to increase its homogeneity is carried out using preparative electrophoresis, gel filtration, chromatography, including affinity and others.

 The aim of the invention is the additional purification of commercial BSA preparations initially isolated from plasma, which is achieved by enzymatic proteolysis of extraneous proteins using trypsin, followed by separation of albumin, trypsin and proteolysis products at Sephadex.

The essence of the claimed method for additional purification of BSA is that crystalline trypsin (or equivalent in activity amount of amorphous trypsin) is added to a 1-3% solution of albumin in 01, M bicarbonate buffer (pH 7.8), based on the ratio of protein: enzyme, equal to 50: 1, is maintained at ³⁷ ° C for 1 hour and then the solution was passed through a column of Sephadex, wherein the albumin (M = 69000) comes off the column before trypsin (M = 21000) and proteolysis products. If it is necessary to obtain protein in a free state, dialysis is performed to remove salts and freeze drying. When choosing the optimal conditions for proteolysis (concentration, temperature, pH) are guided by proven data.

 To control the purity of albumin, we performed disk-electrophoresis of a protein solution in a polyacrylamide gel on a PEFA-1 device before and after proteolysis. The pH values of the separating gel and buffer solution were 8.9 and 8.3, respectively, the staining of the protein zones in the gel was carried out with an amide-black dye. It should be noted that at the indicated pH values the trypsin molecules are positively charged (isoelectric point 10.5) and will not migrate in the gel to the positive electrode, which excludes the effect of the enzyme on the results of electrophoresis.

 The drawing shows electrophoregrams of the initial preparation BSA (a) and protein exposed to trypsin (b).

 As can be seen, as a result of treatment with the enzyme, a significant increase in the degree of homogeneity of the protein preparation is achieved.

To compare the proposed method with BSA homogenization prior art thermal denaturation extraneous protein has been performed, which 1% albumin solution in distilled water was incubated at ⁷⁰ ° C until a slightly cloudy, was filtered and electrophoresed. As can be seen from the electrophoregram shown in drawing (c), complete removal of extraneous proteins could not be achieved, however, the intensity of the albumin protein zone decreases, which indicates a partial loss of the useful component.

 An example of a specific purification procedure for a commercial bovine serum albumin preparation.

Equipment:
1. Laboratory thermostat. 2. The device for electrophoresis PEFA-1, TU 5-375-4231-77. 3. Glass column l = 50 cm, d = 1 cm with Sephadex G = 75 (LKV, Sweden).

 Reagents and materials: 1. BSA grade B produced by the Olaine plant, TU 6-09-10-342-75. 2. Trypsin produced by the Leningrad meat processing plant. 3. 0.1 M bicarbonate buffer, pH 7.8. 4. A set of reagents for conducting disk electrophoresis (according to the passport to the device PEFA-1).

Procedure: 1 g of BSA is dissolved in 100 ml of bicarbonate buffer, filtered to remove sediment. A sample of a protein solution is taken and disk electrophoresis is performed in accordance with the procedure described in the passport for the PEFA-1 device (3), the electrophoregram of the initial BSA solution is shown in drawing (a). To a solution of BSA was added 20 mg of trypsin, stirred and incubated at 37 ^{° C} for 1 hour. The electrophoresis was repeated, the results are shown in the drawing (b). To separate trypsin and proteolysis products, the solution is passed through a Sephadex column, the fraction of purified albumin leaves the column first.

 Feasibility or other efficiency. The proposed method for additional protein purification is an effective means of homogenizing commercial preparations of bovine serum albumin.

Claims (1)

METHOD FOR CLEANING BOBYE SERUM ALBUMINE, including plasma precipitation with ethanol at low temperature, changing pH, ionic strength, removing foreign proteins, characterized in that they additionally carry out the proteolysis of impurity proteins with trypsin in bicarbonate buffer, pH 7.8, at 37 ^° C and bovine serum albumin is separated by gel filtration through Sephadex G-75.

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