| 20026277 |
Zhang L, Liu X, Wang C, Liu X, Cheng G, Wu Y: Expression, purification and direct eletrochemistry of cytochrome P450 6A1 from the house fly, Musca domestica. Protein Expr Purif. 2010 May;71(1):74-8. Epub 2009 Dec 21.
A plasmid (pCW) was modified to code for the complete sequence of house fly (Musca domestica) cytochrome P450 6A1 (CYP6A1) with only the second amino acid changed in the N-terminal portion and this plasmid was used to express the enzyme CYP6A1 in Escherichia coli cells. With the addition of delta-aminolevulinic acid and FeCl (3) to the culture, the enzyme was produced at a level about 0.25 micromol L (-1) (15mgL (-1)) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was characterized and the content of CYP6A1 in each fraction was determined by the spectroscopic method. A nearly homogenous CYP6A1 was obtained by purification with a combination of DEAE Sepharose fast flow and hydroxyapatite chromatography. Direct electrochemistry of CYP6A1 in a didodecyldimethylammonium bromide (DSAB) film on an edge-plane pyrolytic graphite electrode (EPG) has been obtained and the catalytic activity of the enzyme to aldrin has been demonstrated by the cyclic voltammetry. |
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