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

• Published in: Journal of Chromatography A Vol. 848; no. 1; pp. 149 - 159
• Main Authors: Schwaneberg, U, Sprauer, A, Schmidt-Dannert, C, Schmid, R.D
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 02.07.1999; Elsevier
• Subjects: Bacillus megaterium - enzymology; Biological and medical sciences; Biotechnology; Calibration; Chromatography, Ion Exchange - methods; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - isolation & purification; Cytochromes; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Protein engineering; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Spectrophotometry, Ultraviolet; Proteins; Cytochromes; Preparative chromatography; Hemoprotein; Purification; Bacillales; Microbial origin; Cytochrome P450; Anion exchanger; Bacteria; Recombinant protein; Bacillaceae; Bacillus megaterium; Ion exchange chromatography
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00457-4

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 ÄKTA explorer 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.