SU810716A1 - Method of separating proteins and nucleic acids - Google Patents

Description

The invention relates to the production of biochemical preparations and can be used in enterprises receiving enzymes and nucleic acids from biological raw materials.

The source of proteins and nucleic acids is often the cells of microorganisms.

Widely known and well-developed methods for the isolation of proteins (eg, enzymes) and nucleic acids from biological raw materials. Since both proteins and nucleic acids are simultaneously present in the cell mass (or extract prepared from cells), the issue of isolating certain biopolymers is inextricably linked with their separation.

However, currently existing methods for isolating one class of biopolymers from cell extracts are based on the denaturation of another class.

In particular, from the literature methods, the most efficient method for isolating proteins is that the cell extract obtained after separation of the membranes by high-speed centrifugation is treated with protamine sulfate to remove ribosomes and nucleic acids, proteins are fractionated in various concentrations of ammonium sulfate, and liquid is further purified by chromatography and gel filtration [1]. Moreover, one of the biosynthesis products (nucleic acids) becomes a waste product.

At the same time, to obtain nucleic acids, in particular transfer ribonucleic acids (tRNAs). the cell extract to remove proteins is treated with phenol and further purification of TPH1 \ 10 is carried out by liquid chromatography [2].

When using these methods, proteins or nucleic acids are waste products, and a deficient protamine sulfate preparation is used to remove nucleic acids.

The aim of the invention is a more complete use of biological raw materials and the prevention of denaturation of proteins and nucleic acids of the cell extract with 20 separation.

This goal is achieved by the proposed method for the separation of proteins and nucleic acids contained in a cell extract after removing 25 cell membranes and ribosomes, which consists in ion-exchange chromatography of the extract on diethylaminoethyl cellulose in a salt concentration gradient: NH ₄ C1, or NaCJ, or KC1 from 0.02 to 0 , 3 M in 30 buffer solutions with a pH of 7.1–7.5, and then NaCl from 0.3 to 0.7 M in a Ha-acetate buffer pH 5.0–5.5.

Significant differences of the method are the implementation of ion-exchange chromatography of the cell extract on diethylaminoethyl cellulose and the indicated conditions for its implementation.

In the implementation of the method, a cell extract is usually used, obtained after removal of the cell membranes by high-speed centrifugation and ribosomes - ’ultracentrifugation (105000 g).

Example 1. 500 g of the bacterial mass of Escherichia coli strain M.RE-600, disintegrated by rubbing with aluminum oxide, disintegrated biomass suspended in 800 ml of buffer, pH 7.5, containing 0.02 M ammonium chloride, precipitated cell debris and alumina high-speed centrifugation at 27,000 g and then ribosomes are precipitated by ultracentrifugation at 105,000 g. The supernatant after separation of the ribosomes is applied to a column with DEAE cellulose, equilibrated with a buffer, pH 7.5, containing 0.02 M ammonium chloride. Protein elution is carried out in a concentration gradient of ammonium chloride from 0.05 to 0.3 7Λ. Then DEAE cellulose is balanced with 0.05 M sodium acetate buffer, pH 5.0, containing 0.3 M sodium chloride and tRNA is eluted by increasing the concentration of sodium chloride to 0.7 M.

The elongation factor G (EF-G) in the amount of 220 mg is isolated in pure form from the protein fraction. tRNA yield is 1 g.

Note]) 2. 100 g of cell mass of E. coli, M RE-600 infected with bacteriophage T4 amb No. 82, are suspended in 300 ml of 0.01 M Tris HCI buffer, pH 7.6, containing 0.001 M magnesium chloride, and 0.01 M mercaptoethanol (buffer A). Cells are destroyed by ultrasound. Cell debris was pelleted by centrifugation in a rotor (Beckman) at 35,000 rpm for 3 hours. The supernatant was applied to a 5x30 cm column with DEAE cellulose at a rate of 250 ml / h. After washing the column with 1.5 L of buffer A until absorption disappeared at a wavelength of 280 nm, proteins elute in a linear gradient of 0.02 and 0.3 M sodium chloride in the buffer. A (total volume of gra4 diengue 6 l). The DNA and RNA ligase fractions are determined by complexation with [ ³ H] ATP. The DNA ligase fraction is further purified by chromatography on phosphocellulose 5 and hydroxylapatite. The yield is 21.8% of the activity in the crude extract. The RNA ligase fraction is further purified by chromatography on hydroxylapatite and gel filtration on ASA-44 ultragel. Yield 10 is 36.5% of the content in the crude extract.

After elution of the DNA and RNA ligase, the DEAE cellulose column was equilibrated with 0.05 M sodium acetate buffer, pH 15 5.0, containing 0.3 M sodium chloride. tRNA is eluted with the same buffer containing 0.7 M sodium chloride. The output of tRNA is 50 ml.

Claims (2)

20 Forms of invention 1. A method for separation of proteins and nucleic acids contained in the cell extract after cell removal and notation ²⁵ lochek ribosomes, characterized in that, in order to prevent denaturation of proteins or nucleic acids, and a more complete use of biological sy Darya _30, the separation is carried out by ion exchange chromatography diethylaminoethyl cellulose in a gradient with the concentration of salts: ammonium chloride, or sodium chloride, or potassium chloride from 0.02 to ₃₅ 0.3 M in buffer solutions with a pH of 7.1-7.5, and then sodium chloride from 0.3 to 0 , 7 M in n atrium acetate buffer pH 5.0-5.5. 2. The method according to π. 1, ₄₀ characterized in that the cell extract after removal of the high speed centrifugation of cell membranes and ribosomes - ultracentrifugation (105 000 g).