CS199122B1 - Method for the isolation of proteins - Google Patents

Description

The subject of PV relates to the isolation of proteins from biological material having affinity for nucleic acids.

Exactly the stage of enzymes and proteins involved in nucleic acid metabolism in living systems is to be obtained in the Fourth State. Until now, general separation methods have been used to isolate proteins which utilize differences in their molecular weights and ionogenic properties. These methods are time-consuming (lasting from days to weeks), requiring sophisticated instrumentation and high professional qualifications. The protracted operation results in reduced yields of enzyme activities, often related to the loss of their native structure.

These drawbacks are overcome by the isolation method of the present invention, which utilizes the ability of these proteins to specifically bind to a chromatographic ion exchanger consisting of deoxyribonucleic acid bound to polyethyleneimine-carboxymethylcellulose. The use of this ion exchanger for protein affinity chromatography is to remove nucleic acids from biological material.

Example δ. 1

Isolation of RNA-polymeresis from Streptomyces aureofacies.

199 122 g of washed aureofaciens ea cells disintegrate in 20 ml of 0.05M Tris-hydroxymethyl-aminoethane pH 7.9, 0.1 ml of chalatone III and centrifuged at 30,000g for 2 hours at 2 ° C. The supernatant ea mixed with 5g of polyetalenimine-carboxymethylcellulose and added an amount of potassium chloride to give a final concentration of 0.3M. Uiaša ea 1/2 hour and centrifuged at 30,000g. 20 ml of potassium chloride-free buffer was added to the eupernatant and loaded onto a 2 x 5 cm column at a rate of 3 ml / h. From this column, it is eluted with a buffer solution in which the ionic strength is gradually increased by 0.3M, 0.5M, 1M, 2M drapeic chloride. The active enzyme was eluted in fraction with 1M and 2M potassium chloride. In both * 2 cases, the degree of purification is of the order of 5.10 and the enzyme function is fully DNA dependent. The entire purification process takes less than 36 hours.

Example 2I. 2

Isolation of restriction endodezoxyribonuclease Sau I. from Streptomyces aureofaciens.

To 10 g of pre-washed cells from S. aureofaciens was added 20 ml of 0.01 M Trie-hydroxymethyl-aminomethane buffer. After disintegration, the mixture is centrifuged at 105 ° C for 2 hours. at 2 ° C. The whole mixture is loaded onto a 2 x 10 cm chromatography column and eluted sequentially with 0.01 M phosphate buffer containing 0.1 µM chelatone III, 1 mM 2-mercaptoethanol and 108 glycerin. The fraction containing the restriction endonuclease was eluted from the column with 0.1 M potassium chloride in said buffer. This fraction was loaded onto a 1 x 8 cm column with a polyethyleneimine carboxymethylcellulose deoxyribonucleic acid coating and eluted sequentially with 0.1, 0.3, 0.5 M potassium chloride. The active enzyme was eluted in fraction with 0.5 M potassium chloride. The enzyme was devoid of non-specific nucleases.

In this way, various proteins can be isolated from biological materials having affinity for nucleic acids. These enzymes are widely used in molecular biology experiments and genetic engineering.

Claims (1)

OBJECT OF THE INVENTION Protein isolation process characterized in that the proteins bind to a deoxyribonucleic acid-polyethyleneimine-carboxymethylcellulose ion exchanger from which they are released by an ionic strength of 0.1 to 1.5 M at a pH of 7.0-9.2.