Expression, purification and direct eletrochemistry of cytochrome P450 6A1 from the house fly, Musca domestica.
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.
Zhang
Li
L
Key Lab for Biotechnology of National Commission for Nationalities, College of Life Science, The South Central University for Nationalities, Wuhan 430074, PR China.
Liu
Xuequn
X
Wang
Chuntai
C
Liu
Xinqiong
X
Cheng
Gang
G
Wu
Yunhua
Y
D000452:Aldrin / Q000494:pharmacology; D000818:Animals; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism*; D017886:Durapatite; D004563:Electrochemistry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D006793:Houseflies / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose; D013057:Spectrum Analysis
D011994:Recombinant Proteins; C097574:sepharose fast flow SP; D012685:Sepharose; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D000452:Aldrin