An efficient single-step purification protocol for recombinant cytochrome P450 BM-3 from Bacillus megaterium, expressed in E. coli, was developed. Functional crude protein was obtained by disintegrating induced E. coli DH5α and removing cell debris by centrifugation. After investigating different anion-exchange matrices, elution salts and the elution procedures involving an AKTAexplorer system, adsorption of the crude extract from lysed E. coli to Toyopearl DEAE 650M anion exchanger, followed by a two-step elution using NaCl, proved sufficient to isolate almost pure protein without inactivation (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 further optimization to a 6-l production-scale column containing Toyopearl DEAE 650M anion exchanger. Copyright (C) 1999 Elsevier Science B.V.
Bibliographical noteFunding Information:
This work was supported by the Federal Ministry of Education, Research and Technology (BMBF (grant ZSP B3.3U), Bonn, Germany and by BASF AG, Ludwigshafen, Germany.
Copyright 2007 Elsevier B.V., All rights reserved.
- Preparative chromatography