title: Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems.
abstract: Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.
authors.affiliation: Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt/Main, Max-von-LaueStrasse 9, D-60438 Frankfurt/Main, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Schwarz
authors.forename: Friederike
authors.initials: F
authors.lastname: Junge
authors.forename: Florian
authors.initials: F
authors.lastname: Durst
authors.forename: Nadine
authors.initials: N
authors.lastname: Frölich
authors.forename: Birgit
authors.initials: B
authors.lastname: Schneider
authors.forename: Sina
authors.initials: S
authors.lastname: Reckel
authors.forename: Solmaz
authors.initials: S
authors.lastname: Sobhanifar
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D002457:Cell Extracts; D002474:Cell-Free System; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism*; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D003902:Detergents; D008565:Membrane Proteins