title: Co-translational stabilization of insoluble proteins in cell-free expression systems.
abstract: Precipitation, aggregation, and inclusion body (IB) formation are frequently observed problems upon overexpression of recombinant proteins. The open accessibility of cell-free reactions allows addressing such critical steps by the addition of protein stabilizers such as chemical chaperones or detergents directly into the expression reactions. This approach could therefore reduce or even prevent initial protein precipitation already in the translation environment. The strategy might be considered to generally improve protein sample quality and to rescue proteins that are difficult to refold from IBs or from aggregated precipitates. We describe a protocol for the co-translational stabilization of difficult proteins by their expression in the presence of supplements such as alcohols, poly-ions, or detergents. We compile potentially useful compounds together with their recommended stock and working concentrations. Examples of screening experiments in order to systematically identify compounds or compound mixtures that stabilize particular proteins of interest are given. The method can primarily be considered for the production of unstable soluble proteins or of membrane proteins containing larger soluble domains.
authors.affiliation: Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Max-von-Laue-Str. 9, Frankfurt/Main, 60438, Germany.
authors.forename: Lei
authors.initials: L
authors.lastname: Kai
authors.forename: Erika
authors.initials: E
authors.lastname: Orbán
authors.forename: Erik
authors.initials: E
authors.lastname: Henrich
authors.forename: Davide
authors.initials: D
authors.lastname: Proverbio
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology*; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins