Quantitative whole-cell cytochrome P450 measurement suitable for high-throughput application.
The recombinant expression of cytochrome P450 enzymes involved in drug metabolism is of interest to the pharmaceutical and biotechnological industries due to the versatile catalytic properties of these enzymes. Accurate quantification of cytochrome P450 enzymes expressed in bacterial culture generally depends on disruption and fractionation of cells to prepare membranes for spectral analysis. Although whole-cell methods for spectral determination have been reported, problems with poor reproducibility and low signal-to-noise ratio confound the use of such techniques where P450 hemoprotein expression levels are relatively low, such as in cultures of certain mammalian forms. In particular, interference from bacterial hemoproteins often obscures the P450 peak. In the current study, the combination of culture concentration, incubation under microaerobic conditions, and a modified method of baseline correction enabled reproducible quantification of cytochrome P450s in whole cells. This whole-cell method is well suited to high-throughput application, as large sets or libraries of enzymes can be expressed in parallel and relative expression levels measured without downstream cell processing.
Johnston
Wayne A
WA
Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, St Lucia, Brisbane, Australia. w.johnston1@uq.edu.au
Huang
Weiliang
W
De Voss
James J
JJ
Hayes
Martin A
MA
Gillam
Elizabeth M J
EM
D000332:Aerobiosis / Q000502:physiology; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D019151:Peptide Library; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013057:Spectrum Analysis
D019151:Peptide Library; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System