Bacterial expression of membrane-associated cytochrome P450s and their activity assay in nanodiscs

Demetrios Maroutsos, Hannah Huff, Aditi Das

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Eukaryotic membrane bound cytochrome P450s are expressed in bacterial systems to produce large yields of catalytically active protein for structure function studies. Recently, there have been several instances of expressing eukaryotic membrane bound CYPs in bacteria after making various modifications to both the N-terminus membrane binding domains of the protein and to noncontiguous F-G membrane binding loop that is also implicated in substrate binding. These modifications have been shown not to disturb the function of the protein of interest. The major factors that have been key to express the membrane bound cytochrome P450s in bacteria have been the following: (a) exon optimization (b) selection of the appropriate vector and host strain, and (c) growth and expression conditions with respect to temperature and speed of shaking the media flask. Herein, we describe methods to express and purify eukaryotic membrane bound cytochrome P450s. We also describe the measurement of the activity of the cytochrome P450 expressed by taking the example of cytochrome P450 2J2, the primary P450 expressed in the human heart and CYP725A4, the primary cytochrome P450 expressed in the first step of taxol synthesis. Additionally, we discuss the pros and cons of the different modifications done in order to express the membrane bound cytochrome P450s.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages47-72
Number of pages26
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1927
ISSN (Print)1064-3745

Fingerprint

Cytochromes
Membranes
Cytochrome P-450 Enzyme System
Bacteria
Paclitaxel
Exons
Carrier Proteins
Membrane Proteins
Proteins
Temperature
Growth

Keywords

  • Activity assay
  • CYPs
  • Cytochrome P450
  • His-Tags
  • N-terminal modification
  • Protein purification

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Maroutsos, D., Huff, H., & Das, A. (2019). Bacterial expression of membrane-associated cytochrome P450s and their activity assay in nanodiscs. In Methods in Molecular Biology (pp. 47-72). (Methods in Molecular Biology; Vol. 1927). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9142-6_5

Bacterial expression of membrane-associated cytochrome P450s and their activity assay in nanodiscs. / Maroutsos, Demetrios; Huff, Hannah; Das, Aditi.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 47-72 (Methods in Molecular Biology; Vol. 1927).

Research output: Chapter in Book/Report/Conference proceedingChapter

Maroutsos, D, Huff, H & Das, A 2019, Bacterial expression of membrane-associated cytochrome P450s and their activity assay in nanodiscs. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1927, Humana Press Inc., pp. 47-72. https://doi.org/10.1007/978-1-4939-9142-6_5
Maroutsos D, Huff H, Das A. Bacterial expression of membrane-associated cytochrome P450s and their activity assay in nanodiscs. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 47-72. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-9142-6_5
Maroutsos, Demetrios ; Huff, Hannah ; Das, Aditi. / Bacterial expression of membrane-associated cytochrome P450s and their activity assay in nanodiscs. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 47-72 (Methods in Molecular Biology).
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