Measurement of P450 difference spectra using intact cells

Johnston, Wayne A. and Gillam, Elizabeth M. J. (2013). Measurement of P450 difference spectra using intact cells. In Ian R. Phillips, Elizabeth A. Shepherd and Paul R. Ortiz de Montellano (Ed.), Cytochrome P450 protocols 3rd ed. (pp. 189-204) New York, NY, United States: Humana Press. doi:10.1007/978-1-62703-321-3_17

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Author Johnston, Wayne A.
Gillam, Elizabeth M. J.
Title of chapter Measurement of P450 difference spectra using intact cells
Title of book Cytochrome P450 protocols
Place of Publication New York, NY, United States
Publisher Humana Press
Publication Year 2013
Sub-type Research book chapter (original research)
DOI 10.1007/978-1-62703-321-3_17
Series Methods in Molecular Biology
Edition 3rd
ISBN 9781627033206
ISSN 1064-3745
Editor Ian R. Phillips
Elizabeth A. Shepherd
Paul R. Ortiz de Montellano
Volume number 987
Chapter number 17
Start page 189
End page 204
Total pages 16
Total chapters 25
Collection year 2014
Language eng
Formatted Abstract/Summary
Whole-cell assays provide a rapid means of determining expression and substrate binding for cytochrome  P450 enzymes expressed heterologously in Escherichia coli and, potentially, other host cells. Such assays are  particularly useful for screening large libraries of mutant P450s, where rapid, high-throughput assays are  needed for fi rst-tier screens that can, firstly, quantify any P450 form independent of P450 subfamily and,  secondly, suggest possible ligands before more labor-intensive direct measurement of substrate turnover. Whole-cell spectral techniques are derived from methods that have been used for a long time to study  P450s in microsomal or other subcellular fractions (Omura T and Sato R, J Biol Chem 239:2370–2378, 1964; Schenkman JB et al., Biochemistry 11:4243–4251, 1972), but recent studies have detailed important modi fi cations which allow quantitative results to be obtained in whole cells (Otey CR, Methods in Molecular Biology, vol. 230, Humana, Totowa, NJ, pp. 137–139, 2003; Johnston WA et al., J Biomol Screen 13:135–141, 2008). A general method is presented here for the measurement of difference spectra on recombinant P450 cultures that can be applied to both carbon monoxide and any number of alternative ligands that alter the characteristic spectral signature of P450s.
Keyword Cytochrome P450
Directed evolution
Escherichia coli
Difference spectroscopy
High-throughput screening
DNA shuffling
Q-Index Code BX
Q-Index Status Confirmed Code
Institutional Status UQ

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Created: Mon, 15 Apr 2013, 21:40:11 EST by Elizabeth Gillam on behalf of School of Chemistry & Molecular Biosciences