title: Affinity purification of recombinant human cytochrome P450s 3A4 and 1A2 using mixed micelle systems.
abstract: Recombinant cytochrome P450 (CYP or P450) enzymes are useful for drug metabolism research and thereby many expression and purification systems have been developed. Here, we provide a method for the purification of human P450s 3A4 and 1A2 expressed in Escherichia coli using mixed micelles containing anionic phospholipids. This method does not require any protein-tagging system for protein isolation and has a further advantage that the purification is concomitantly conducted with reconstitution of the enzymes into a phospholipid environment, which is crucial for the catalytic activity assay of P450 enzyme. This method may also be applied to high-throughput catalytic assays of the enzymes because the purification procedures can be undertaken in a 96-well plate.
authors.affiliation: College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea. Electronic address: thahn@chonnam.ac.kr.
authors.forename: Taeho
authors.initials: T
authors.lastname: Ahn
authors.affiliation: College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea.
authors.forename: Chun-Sik
authors.initials: CS
authors.lastname: Bae
authors.affiliation: School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
authors.forename: Chul-Ho
authors.initials: CH
authors.lastname: Yun
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008823:Micelles; D010743:Phospholipids / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D008823:Micelles; D010743:Phospholipids; D011994:Recombinant Proteins; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human
keywords: Affinity purification; Catalytic activity; Cytochrome P450; Mixed micelle