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Affinity purification of recombinant human cytochrome P450s 3A4 and 1A2 using mixed micelle systems
Ist Teil von
Protein expression and purification, 2014-09, Vol.101, p.37-41
Ort / Verlag
United States: Elsevier Inc
Erscheinungsjahr
2014
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
•We provide a purification method for P450s 3A4 and 1A2 using mixed micelles.•Mixed micelles increase a fold-purification of the recombinant enzymes.•This method enables one-step purification of the enzymes without an affinity-tag.•This method may also be applied to high-throughput catalytic assays of the enzymes.
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.