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
- Cell-free expression
- Escherichia coli
- Membrane protein
- X-ray structural analysis
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Received and accepted: October 29, 2009
Published online: February 17, 2010
Number of Print Pages : 7
Number of Figures : 5, Number of Tables : 1, Number of References : 19
Journal of Molecular Microbiology and Biotechnology
Vol. 18, No. 2, Year 2010 (Cover Date: April 2010)
Journal Editor: Saier Jr. M.H. (La Jolla, Calif.)
ISSN: 1464-1801 (Print), eISSN: 1660-2412 (Online)
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