P450 monooxygenase in biotechnology - I. Single-step, large-scale purification method for cytochrome P450BM-3 by anion-exchange chromatography

An efficient single-step purification protocol for recombinant cytochrome P 450 BM-3 from Bacillus megaterium, expressed in E. coli, was developed. Fun ctional crude protein was obtained by disintegrating induced E. coli DH5 al pha and removing cell debris by centrifugation. After investigating differe nt anion-exchange matrices, elution salts and the elution procedures involv ing an AKTAexplorer system, adsorption of the crude extract from lysed E. c oli to Toyopearl DEAE 650M anion exchanger, followed by a two-step elution using NaCl, proved sufficient to isolate almost pure protein without inacti vation (up to 93% P450 BM-3 content) in yields that ranged between 79-86%. The purification method could be scaled up 1500-fold and higher without fur ther optimization to a 6-1 production-scale column containing Toyopearl DEA E 650M anion exchanger. (C) 1999 Elsevier Science B.V. All rights reserved.