title: Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies.
abstract: The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments.
authors.affiliation: Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Max-von-Laue-Str. 9, 60438, Frankfurt/Main, Germany.
authors.forename: Ralf-Bernhardt
authors.initials: RB
authors.lastname: Rues
authors.affiliation: Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Max-von-Laue-Str. 9, 60438, Frankfurt/Main, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Henrich
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.
authors.forename: Coilin
authors.initials: C
authors.lastname: Boland
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.
authors.forename: Martin
authors.initials: M
authors.lastname: Caffrey
authors.affiliation: Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Max-von-Laue-Str. 9, 60438, Frankfurt/Main, Germany. fbern@bpc.uni-frankfurt.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D002474:Cell-Free System; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D017510:Protein Folding; D012995:Solubility; D058615:Synthetic Biology
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins
keywords: G-protein-coupled receptors; Lipid screening; Membrane protein crystallization; Membranes; Nanodiscs; Synthetic biology