OVEREXPRESSION AND PURIFICATION OF THE TRIMERIC ASPARTATE TRANSCARBAMOYLASE FROM BACILLUS-SUBTILIS

A procedure has been developed for the overexpression and purification of milligram quantities of the Bacillus subtilis aspartate transcarba moylase. The plasmid pEK171, carrying the B. subtilis pyrB structural gene under the control of the Escherichia coli pyrBI promoter, was tra nsformed into the E, coli strain EK1104 and the enzyme overexpressed t o approximately 50% of total soluble protein under extreme derepressio n of the pyrimidine pathway. The enzyme was subsequently purified by m eans of ammonium sulfate fractionation, anionic exchange chromatograph y using Q-Sepharose Fast Flow resin, negative chromatography on Matrex Gel Red A agarose, and hydrophobic interaction chromatography using M atrex Phenyl Cellufine, The purification yields approximately 60 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the o verexpressed enzyme indicated that it had kinetic properties very simi lar to those of the enzyme purified from B. subtilis cells. (C) 1995 A cademic Press, Inc.