SU1638162A1 - Method of isolation of reverse transcriptase from escherichia coli - Google Patents

Abstract

The invention relates to biotechnology and can be used to obtain an enzyme that transforms genetic information from RNA into DNA molecules. The aim of the invention is to increase the degree of purification of the reverse transcriptase from E coli. Reverse transcriptase production involves homogenization of E coli MRE-600 cells, extraction of the protein with a buffer solution, fractionation in a two-phase PEG-dextran system, followed by purification using column chromatography on phosphorus cellulose, DEAE-cellulose, Sephadex G-100, and Bio-Receptor chromatography 70. The degree of purification is 1358, the specific activity of the preparation is 57000 units / mg, the yield for activity is 2.8%, the yield for protein is 0.002%. 1 tab

Description

The invention relates to biotechnology and can be used to obtain an enzyme that transforms genetic information from RNA into DNA molecules.

The aim of the invention is to increase the degree of purification of reverse transcriptase from E. colt.

Reverse transcriptase production involves homogenization of E. coli MRE 600 cells, extraction of the protein with a buffer solution, fractionation in a two-phase polyethylene glycol-dextran system, followed by chromatographic purification, including sequential chromatography on phosphocellulose, diethylaminoethyl cellulose, Sephadex G-100, and Bio-Rex 70 carrier.

The degree of purification and yield at each stage are given in the table.

The method is illustrated by examples.

Example Obtaining Reverse Transcriptase from E. coli

1. The destruction of bacterial cells and obtaining a coarse extract.

To 1.5 kg of MRE-600 biomass of the early logarithmic growth phase, 750 ml of an aqueous solution of lysozyme with a concentration of 2 mg / ml is added. The suspension is titrated with a saturated solution of Tris- (oxymethyl) -aminomethane to pH 8.3, incubated for 30 minutes at 5-7 ° C, the mixture is frozen in liquid nitrogen and destroyed in a mechanical homogenizer at

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8000 rpm for 10 min. 3000 ml of 0.05 M Tris-HCl, pH 8.3 is added to the obtained disrupted cell powder, and extraction is carried out with a mechanical stirrer for 1.5 minutes. The extract is clarified by centrifugation (60 min, 9000 rpm).

The protein yield at this stage is 72 g; the specific activity of the enzyme is 42 ea / mg.

2, Polyethylene glycol-dextran fractionation in a two-phase system.

1095 ml of 30% polyethylene glycol solution (PEG, mol. 20,000 D) and 200 ml of 20% dextran T-500 solution are added to 3300 ml of the obtained crude extract. The resulting mixture is stirred for 30 minutes using a mechanical stirrer. Then centrifuged for 40 min at 9000 rpm. The upper phase is discarded, and a solution containing 0.4 M KCl, 50 mM Tris-HCl (pH 8.3) and 6% PEG solution (mol, wt. 20,000 D) is added at the rate of 8 vol. of this solution on 1 of the lower phase. After 30 minutes of vigorous stirring, the phases are again separated by centrifugation (40 minutes, 9000 rpm) and the upper phase is discarded. To the lower phase add a solution containing 4M KCI and 6% PEG (mol.mash. 6000D) in 50 mM Tris-HCl, pH 7.3, at the rate of 3 v of this solution per

About the lower phase and mix with a mechanical stirrer for 40 minutes. After 40 minutes of centrifugation at 9000 rpm, the upper phase is then decanted and the contained material is cleaned.

The protein yield of 12.7 g; specific activity of 76 ea / mg. The yield on the activity of 32%.

3. Chromatography on phosphocellulose P-11.

The protein material obtained in the previous step is freed from polyethylene glycol, nucleic acid fragments, other low molecular weight impurities / and also excess salt by passing through a column with Sephadex G-25 (coarse, V 8 L), equilibrated with 50 mM Tris-HCl, pH 7, 3, containing 50 mM CS, and applied to a column with PI1 phosphocellulose (V 400 ml), equilibrated with a buffer solution of the same composition. Separation is carried out in a linear gradient of KS 0.05-0.5 M in 50 mM Tris-HCI, pH 7.3 (total gradient volume 4 l). The fractions containing reverse transcriptase activity are combined and protein precipitation is carried out by dialysis against a saturated solution of ammonium sulfate, pH 7.5. The protein precipitate is collected by centrifugation (9000 rpm, 45 min). h

The protein yield at this stage is 120 mg specific activity of 2300 ea / mg.

4. Chromatography on diethylaminoethyl cellulose DE-52,

The protein material obtained at the previous stage is desalted on a Sephadex G-50 column (average V 100 ml), equilibrated with 0.05 M Tris-HCl, pH 7.3, containing 50 mM KCI, and applied to a column with

diethylaminoethylcellulose DE-52 (V 100 ml), balanced with a buffer solution of the same composition. Chromatographic separation is carried out in a linear KCI gradient of 0.05-0.5 M Tris-HCI, pH 7.3.

The total volume of the gradient is 1.0 l. Chromatographic fractions containing a protein with reverse transcriptase activity and not containing nucleases are pooled, the protein is precipitated by dialysis against a suspension of ammonium sulfate, as in step 3, and the protein precipitate is collected by centrifugation.

Protein yield 8.7 mg; specific activity - 12000 EA / mg.

5. Gelfiltration on Sephadex G-100.

The protein precipitate is dissolved in a volume of 1-2 ml of 0.05 M Tris-HCl, pH 7.3, containing 0.05 M KCl, the solution is clarified by centrifugation, and the protein material is applied onto

column with Sephadex G-100 (ultra-thin, V 100 ml, column size: diameter 1 cm, height 70 cm), Application and gel filtration rate 12-14 ml / h. The eluent is 0.05 M Tris-HCI. The fractions containing the protein with reverse transcriptase activity and not containing nucleases are combined and the next purification step is carried out.

The protein yield at this stage is 6 mg; specific activity of 15,000 EA / mg.

6. Chromatography on Bio-Rex 70.

The proteinaceous material obtained in the previous stage was applied to a column (V 2 ml) with Bio-Rex 70, equilibrated with 0.05 M Tris-HCl, pH 7.3, containing

0.05 M KCI, at a rate of 4 ml / h. Separation is carried out in a linear KCI gradient of 0.05-0.5 M Tris-HCl, pH 7.3 (total gradient volume 20 ml) at a rate of 3 ml / h. Fractions containing reverse transcriptase,

unite.

Protein yield 1.5 mg (concentration about 0.5 mg / ml); specific activity 57000 EA / mg.

The enzyme is stored as a frozen solution at -20 ° C without loss of activity for at least one year.

Claims (1)

Formula of the Invention A method of obtaining reverse transcriptase from Escherichia coll MRE-600, including the destruction of bacteria cells, protein extraction with a buffer solution, fractionation with a stepwise increase in salt concentration in a two-phase polyethylene glycol-dextran system and purification by column chromatography, characterized in that increasing the degree of purification, fractionation in the two-phase system of polyethylene glycol-dextran is carried out with a stepwise increase in the concentration of CG to 3 M, and purification by column chromatography is performed sequentially on phosphocellulose P-I in a linear gradient of KCl concentration from 0.05 to 0.5 M in buffer pH 7.3, on DEAE DE-52 cellulose in a linear KCI concentration gradient from 0.05 to 0.5 M in a low ionic strength buffer, pH 7.3, on G-100 sephadex and on a Bio-Rex 70 carrier in a linear concentration gradient KCI from 0.05 to 0.5 M in low ionic strength buffer, pH 7.3.