title: High-level cell-free production of membrane proteins with nanodiscs.
abstract: This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.
authors.affiliation: Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Frankfurt/Main, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Roos
authors.forename: Lei
authors.initials: L
authors.lastname: Kai
authors.forename: Stefan
authors.initials: S
authors.lastname: Haberstock
authors.forename: Davide
authors.initials: D
authors.lastname: Proverbio
authors.forename: Umesh
authors.initials: U
authors.lastname: Ghoshdastider
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.forename: Slawomir
authors.initials: S
authors.lastname: Filipek
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D001483:Base Sequence; D002474:Cell-Free System; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D005285:Fermentation; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012995:Solubility
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D012321:DNA-Directed RNA Polymerases