title: Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.
abstract: Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.
authors.affiliation: Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Focke
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University , Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hein
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University , Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Hoffmann
authors.affiliation: Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Matulef
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University , Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University , Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University , 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239, United States.
authors.forename: Francis I
authors.initials: FI
authors.lastname: Valiyaveetil
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002474:Cell-Free System; D018360:Crystallography, X-Ray; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D015221:Potassium Channels / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D015221:Potassium Channels; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel