Quantitative whole-cell cytochrome P450 measurement suitable for high-throughput application
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Description
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.
Impact and interest:
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ID Code: | 241525 | ||
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Item Type: | Contribution to Journal (Journal Article) | ||
Refereed: | Yes | ||
ORCID iD: |
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Measurements or Duration: | 7 pages | ||
Keywords: | Cytochrome P450, Escherichia coli, Heterologous expression, High-throughput screening, Whole-cell spectra | ||
DOI: | 10.1177/1087057107312780 | ||
ISSN: | 1087-0571 | ||
Pure ID: | 139970788 | ||
Divisions: | Current > QUT Faculties and Divisions > Faculty of Science Current > Schools > School of Biology & Environmental Science |
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Copyright Owner: | © 2008 Society for Biomolecular Sciences | ||
Copyright Statement: | This work is covered by copyright. Unless the document is being made available under a Creative Commons Licence, you must assume that re-use is limited to personal use and that permission from the copyright owner must be obtained for all other uses. If the document is available under a Creative Commons License (or other specified license) then refer to the Licence for details of permitted re-use. It is a condition of access that users recognise and abide by the legal requirements associated with these rights. If you believe that this work infringes copyright please provide details by email to qut.copyright@qut.edu.au | ||
Deposited On: | 17 Jul 2023 03:20 | ||
Last Modified: | 12 Jul 2024 17:10 |
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