title: Labeling of membrane proteins by cell-free expression.
abstract: The particular advantage of the cell-free reaction is that it allows a plethora of supplementation during protein expression and offers complete control over the available amino acid pool in view of concentration and composition. In combination with the fast and reliable production efficiencies of cell-free systems, the labeling and subsequent structural evaluation of very challenging targets, such as membrane proteins, comes into focus. We describe current methods for the isotopic labeling of cell-free synthesized membrane proteins and we review techniques available to the practitioner pursuing structural studies by nuclear magnetic resonance spectroscopy. Though isotopic labeling of individual amino acid types appears to be relatively straightforward, an ongoing critical issue in most labeling schemes for structural approaches is the selective substitution of deuterons for protons. While few options are available, the continuous refinement of labeling schemes in combination with improved pulse sequences and optimized instrumentation gives promising perspectives for extended applications in the structural evaluation of cell-free synthesized membrane.
authors.affiliation: Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany. Electronic address: laguerre@bpc.uni-frankfurt.de.
authors.forename: Aisha
authors.initials: A
authors.lastname: LaGuerre
authors.affiliation: Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.affiliation: Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.affiliation: Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
mesh_terms: D002474:Cell-Free System; D008565:Membrane Proteins / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular
chemical_list: D008565:Membrane Proteins
keywords: Amino acid scrambling; Cell lysates; Cell-free expression; Deuteration; Membrane mimetics; Membrane proteins; NMR spectroscopy; Nanodiscs; Stable isotopes; Synthetic biology