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

U. Schwaneberg, A. Sprauer, C. Schmidt-Dannert, R. D. Schmid

Research output: Contribution to journalArticle

59 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)149-159
Number of pages11
JournalJournal of Chromatography A
Volume848
Issue number1-2
DOIs
StatePublished - Jul 2 1999

Keywords

  • Cytochromes
  • Preparative chromatography
  • Proteins

Fingerprint Dive into the research topics of 'P450 monooxygenase in biotechnology. I. Single-step, large-scale purification method for cytochrome P450 BM-3 by anion-exchange chromatography'. Together they form a unique fingerprint.

  • Cite this