ECO-RI restriction endonuclease prodn. - by submerged culture of E coli SB5 and multistage purificn. of prod.

摘要

ECo-RI restriction endonuclease is prepd. form E.Coli SBS (I) which has no antibiotic resistance but contains ECori. (I) is fed (up to the logarithmic stage) into an aq. nutrient contg. proteins, inorganic salts and glycerol as C- and energy source, with supply of opt. O2-enriched air, at 30-40 degreesC. Submerged culture is initiated. Cellular material (II) is centrifuged out of the reaction system and the soluble supernatant sepd. off. (II) is suspended in an aq. PEMA(10 mM KH2PO4-K2PO4) of pH 7.0; 0.7mM mercapto ethanol and 1 mM EDTA) with 0.1M NaCl and opt. 0.2% nonionic detergent at 0-4 degrees C. Enzymatically active fraction is sepd. by stirring from (II) which is centrifuged and removed. The residual soln. is stirred with cetyl trimethyl ammonium bromide to give a final concn. =1 vol. %, in order to remove residual side activity. The ppte. formed is sepd. and removed. Supernatant is stirred with phosphocellulose. It is then washed with 0.1M NaCl buffer and then elluted with 1M NaCl buffer and then eluted with 1M NaCl buffer. NaCl is removed by dialysis. Prod. is fed through phosphocellulose column and the active fraction eluted using a linear 0.3-0.8M NaCl gradient, and collected in fractions. The active fractions are fed to a hydroxyapatite column washed with 0.1M NaCl buffer and then eluted with a linear 0.01-0.5M phosphate gradient and collected in fractions. The active fractions are concd. to give high purity ECo-RI-restriction endonuclease.