Bacterial restriction endonuclease prodn. - from antibiotic-sensitive Escherichia coli SB5 DSM N0.656

摘要

A new process is claimed for the prodn. of ECoRI restriction endonuclease from Escherichia Coli SB5 DMS No. 686, which has no antibiotic resistance but does have "ECo-RI". In this process, Escherichia coli SB5 is carried to the logarithmic phase under aerobic conditions in an aq. nutrient medium contg. proteins, inorganic salts and glycerine with aeration with air or O2-enriched air at 30-40 degrees C to produce a submerged culture from which the cell mass is centrifuged off. The cell mass is suspended at 0-4 degrees C in an aq. PEMA (10 mM KH2PO4K2HPO, pH 7.0, 0.7 mM mercaptoethanol, 1 mM EDFA) with ca 0.1 M NaCl and, opt., ca 0.2% non-ionic detergent and a part of the enzymatic activity is dissolved from the undisrupted cell mass by stirring with a shear force. The cell mass is centrifuged off and the supernatant is stirred with cetyltrimethylammonium bromide (end concn. 1 vol.%) to destroy undesired activities and the resulting ppte. is sepd. off and discarded. The supernatant is stirred with phosphocellulose, which is then washed with buffer contg. 0.1 M NaCl and eluted with buffer contg. 1 M NaCl. The eluate is dialysed against buffer contg. 0.1 M NaCl, then pumped onto a phosphocellulose column. The enzyme activity is bound to the column and the soln. is discarded. The column is eluted with a linear 0.3-0.8 M NaCl gradient and the eulate is collected in fractions. The fractions are tested, and those with ECo-RI activity are pumped onto a hydroxylapatite column. After washing with 0.1 M NaCl buffer, the column is eluted with a linear 0.01-0.5 M phosphate gradient, the eluate being collected in fractions. The fractions shown by testing to be active are conc. using a membrane tube to give highly pure conc. ECo-RI restriction endonuclease. ECo-RI restriction endonuclease can be used in the prodn. of recombinant DNA, which permits inclusion of new genetic information in bacteria. Appropriate choice of the DNA used can give bacteria capable of producing important natural products. The e. coli. strain used has no antibiotic resistance. It has been deposited in the German Collection of Microorganisms under the No. DSM 656.