Springer Protocols: Abstract: Cell-Free Production of Integral Membrane Proteins on a Preparative Scale

Cell-Free Production of Integral Membrane Proteins on a Preparative Scale

By: Christian Klammt², Daniel Schwarz², Volker Dötsch², Frank Bernhard²

Abstract

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The chapter will focus on the high level cell-free production of integral membrane proteins having multiple transmembrane segments by using an individual coupled transcription/translation system based on an Escherichia coli S30-extract. We describe in detail the setup and optimization of the cell-free expression technique to obtain the maximum yield of recombinant proteins. The protocol can be used for the expression of soluble membrane proteins as well as for their production as a precipitate. In addition, we will provide protocols for the efficient solubilization and reconstitution of membrane proteins directly from the cell-free produced precipitates.

Affiliation(s): (2) Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany

Book Title: In Vitro Transcription and Translation Protocols

Series: Methods in Molecular Biology | Volume: 375 | Pub. Date: May-03-2007 | Page Range: 57-78 | DOI: 10.1007/978-1-59745-388-2_3

Subject: Genetics/Genomics

Key Words: Integral membrane proteins - structural analysis - solubilization - reconstitution - detergent - NMR spectroscopy - amino acid transporter - G-protein coupled receptors - β-barrel proteins - multidrug resistance

References

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