title: P450 monooxygenase in biotechnology. I. Single-step, large-scale purification method for cytochrome P450 BM-3 by anion-exchange chromatography.
abstract: 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 DH5 alpha 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 6-1 production-scale column containing Toyopearl DEAE 650M anion exchanger.
authors.affiliation: Institut für Technische Biochemie, Universität Stuttgart, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Schwaneberg
authors.forename: A
authors.initials: A
authors.lastname: Sprauer
authors.forename: C
authors.initials: C
authors.lastname: Schmidt-Dannert
authors.forename: R D
authors.initials: RD
authors.lastname: Schmid
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology*; D001709:Biotechnology; D002138:Calibration; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
chemical_list: D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System