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Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay

Paper ID Volume ID Publish Year Pages File Format Full-Text
23792 43473 2012 8 PDF Available
Title
Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay
Abstract

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.

► First report on the surface display of a human first-pass metabolizing enzyme. ► Whole cells displaying CYP3A4 were used to convert testosterone. ► Important step towards in vitro studies on human metabolism of drugs.

Keywords
Autodisplay; Human Cytochrome P450 3A4; Biocatalysis; Surface display; Phase-1-metabolism
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Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 161, Issue 2, 15 October 2012, Pages 113–120
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us