Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2

Protein Expr Purif. 2000 Apr;18(3):310-5. doi: 10.1006/prep.1999.1195.

Abstract

An N-terminally modified form of the Arabidopsis NADPH-cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2',5'-ADP-agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 micromol min(-1) mg(-1) and the K(m) for cytochrome c reduction was 15 +/- 2 microM. The purified NADPH-cytochrome P450 reductase was able to support function of CYP79B2.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / chemistry*
  • Base Sequence
  • Chromatography, Affinity
  • Chromatography, Agarose
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / metabolism
  • Cytochrome c Group / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Mixed Function Oxygenases / metabolism
  • Molecular Sequence Data
  • NADH, NADPH Oxidoreductases / chemistry
  • NADH, NADPH Oxidoreductases / isolation & purification*
  • NADH, NADPH Oxidoreductases / metabolism
  • NADPH-Ferrihemoprotein Reductase
  • Oxidation-Reduction
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA

Substances

  • Cytochrome c Group
  • Recombinant Proteins
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • cytochrome P450TYR
  • NADH, NADPH Oxidoreductases
  • NADPH-Ferrihemoprotein Reductase