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1 June 2007 Functional Expression of Mosquito NADPH-Cytochrome P450 Reductase inEscherichia coli
Dolnapa Kaewpa, Soamrutai Boonsuepsakul, Pornpimol Rongnoparut
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Abstract

A complete NADPH-cytochrome P450 reductase (CPR) cDNA was isolated fromAnopheles minimus Theobald mosquitoes by using reverse transcription-polymerase chain reaction-based methods. The complete cDNA contains 2,040 bp encoding the protein of 679 amino acids, with a calculated molecular mass of 77.3 kDa. The deduced amino acid sequence had a typical feature of CPR by possessing conserved domains involved in binding of flavin mononucleotide, flavin adenine dinucleotide, and NADPH cofactors. The complete CPR cDNA was expressed as 6x His-tagged fusion protein in both membrane and cytosolic fractions inEscherichia coli, both fractions contained NADPH-cytochromec reducing activity. The membrane-bound form containing N-terminal membrane anchor was subjected to purification, andKm values were determined for NADPH and cytochromec. The purified CPR enzyme was functionally active, as demonstrated by its ability to support CYP6AA3-mediated metabolism in the reconstituted reaction in vitro. Initial test suggested that CYP6AA3 could play a role in deltamethrin metabolism.

Dolnapa Kaewpa, Soamrutai Boonsuepsakul, and Pornpimol Rongnoparut "Functional Expression of Mosquito NADPH-Cytochrome P450 Reductase inEscherichia coli," Journal of Economic Entomology 100(3), 946-953, (1 June 2007). https://doi.org/10.1603/0022-0493(2007)100[946:FEOMNP]2.0.CO;2
Received: 31 October 2006; Accepted: 10 February 2007; Published: 1 June 2007
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KEYWORDS
Anopheles minimus
CYP6AA3
deltamethrin
kinetics
NADPH-cytochrome P450 reductase
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