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Table of Contents
Vol. 18, No. 2, 2010
Issue release date: April 2010
Section title: Research Article
Editor's Choice -- Free Access
J Mol Microbiol Biotechnol 2010;18:85–91

An Escherichia coli-Based Cell-Free System for Large-Scale Production of Functional Mammalian Membrane Proteins Suitable for X-Ray Crystallography

Nguyen T.A. · Lieu S.S. · Chang G.
Department of Molecular Biology, The Scripps Research Institute, La Jolla, Calif., USA
email Corresponding Author

Geoffrey Chang

Department of Molecular Biology, The Scripps Research Institute

10550 North Torrey Pines Road

La Jolla, CA 92037 (USA)

Tel. +1 858 784 9490, Fax +1 858 784 9985, E-Mail gchang@scripps.edu

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A cell-free expression system using an Escherichia coli extract was adapted for large-scale expression and purification of mammalian membrane proteins. The system was tested with a set of human membrane proteins of different sizes, numbers of transmembrane domains, oligomeric arrangements, and native membrane locations. Tens of milligrams of protein were readily expressed and purified from an overnight cell-free reaction. Both reaction ‘mode A’ (proteins were expressed as precipitant) and ‘mode B’ (proteins were expressed in the presence of mild detergents to keep them soluble) were investigated. The combination of ‘mode B’ and the right detergents, used in the subsequent extraction and purification steps, is critical for obtaining properly folded proteins (CX32 and VDAC1) that can be crystallized and diffracted (VDAC1). The E. coli cell-free system is capable of efficient expression of many mammalian membrane proteins. However, fine-tuning of the system, especially to facilitate proper protein folding, will be required for each specific target.

© 2010 S. Karger AG, Basel

Article / Publication Details

First-Page Preview
Abstract of Research Article

Received: October 29, 2009
Published online: February 17, 2010
Issue release date: April 2010

Number of Print Pages: 7
Number of Figures: 5
Number of Tables: 1

ISSN: 1464-1801 (Print)
eISSN: 1660-2412 (Online)

For additional information: http://www.karger.com/MMB

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