ABSTRACTNADPH-cytochrome P450 oxidoreductase (POR) is a membrane protein in the endoplasmic reticulum of eukaryotic cells. POR is as a key reducing partner for a number of cytochrome P450 proteins involved in different metabolic degradation and signaling pathways. Preparation of the full-length recombinant POR expressed in bacteria has been reported and, typically, involved the use of Triton X-100 detergent for extraction of the overexpressed POR from bacterial membranes. However, extraction efficiency is always relatively low hindering structural studies, particularly—the NMR spectroscopy requiring isotopic enrichment. In this paper, we assessed the effect of a variety of detergents and additives on the efficiency of the membrane-extraction step in POR preparation protocol. We evaluated non-ionic detergents with the variable hydrophobicity (Triton X-100, X-114, and X-405) and structure (Triton X-100, TWEEN-20, Brij-35), a zwitterionic/non-ionic detergent combination (Triton X-100 and CHAPS), as well as a range of alkylamines and polyamines as additives to the conventional extraction buffer containing Triton X-100. None of the detergents or detergent-additive combinations yielded better extraction efficiency than the conventional protocol with the Triton X-100. Lack of variation of the extraction yield allows to hypothesize that the conventional protocol extracts all of the available natively-folded monomeric POR while the remaining fraction is possibly an unfolded aggregated POR, which did not insert in the membranes during expression. We propose that the yield of soluble POR may be increased by a careful optimization of expression conditions while monitoring the distribution of POR between soluble and insoluble fractions in the detergent extraction step.
Amphipol-facilitated Elucidation of the Functional Tetrameric Complex of Full-length Cytochrome P450 CYP2B4 and NADPH-Cytochrome-P450 Oxidoreductase
