Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay.
The cytochrome P450 enzyme system comprises a large group of enzymes catalyzing a broad diversity of reactions and an extensive substrate specificity, which makes them the most versatile known catalysts. CYP3A4 is one of the important human P450 enzymes and involved in the oxidation of a large range of substrates including toxins and pharmaceuticals. Bottlenecks in studying this enzyme include the difficulty in expressing it in a bacterial host, its need for membrane surroundings and the limited substrate accessibility of enzymes expressed within the cell. To circumvent these difficulties, human CYP3A4 was expressed on the outer membrane of Escherichia coli using Autodisplay. Transport of CYP3A4 to the cell surface was monitored by SDS-PAGE and Western blot analysis of outer membrane proteins. Localization on the cell envelope was determined by flow cytometry after immunolabeling, a whole cell ELISA and a protease accessibility assay. A HPLC assay confirmed the catalytic activity of displayed CYP3A4, using testosterone as a substrate. This activity required the external addition of electron supplying enzymes, however surprisingly, we found that the external addition of a heme group was not necessary. Our results indicate that human CYP3A4 can be recombinantly expressed by surface display in a gram-negative bacterium.
Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
Stephanie D
SD
Schumacher
Joachim
J
Jose
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003062:Codon; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006900:Hydroxylation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003062:Codon; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human