Bacterial Expression and Purification of the Arabidopsis NADPH–Cytochrome P450 Reductase ATR2 |
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Authors: | Anna K Hull John L Celenza |
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Institution: | Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts, 02215 |
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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 μmol min−1 mg−1 and the Km for cytochrome c reduction was 15 ± 2 μM. The purified NADPH–cytochrome P450 reductase was able to support function of CYP79B2. |
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