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This version was published on February 1, 2008
Journal of Biomolecular Screening, Vol. 13, No. 2, 135-141 (2008)
DOI: 10.1177/1087057107312780

Quantitative Whole-Cell Cytochrome P450 Measurement Suitable for High-Throughput Application

Wayne A. Johnston

Physiology and Pharmacology, School of Biomedical Sciences, w.johnston1{at}uq.edu.au

Weiliang Huang

Physiology and Pharmacology, School of Biomedical Sciences

James J. De Voss

Chemistry, School of Molecular and Microbial Sciences, University of Queensland, St. Lucia, Australia

Martin A. Hayes

Discovery DMPK&BAC, AstraZenecaR&D, Mölndal, Sweden

Elizabeth M.J. Gillam

Physiology and Pharmacology, School of Biomedical Sciences

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. (Journal of Biomolecular Screening 2008:135-141)

Key Words: cytochrome P450 • heterologous expression • Escherichia coli • whole-cell spectra • high-throughput screening


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