title: Cell-free expression and stable isotope labelling strategies for membrane proteins.
abstract: Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on alpha-helical transmembrane proteins which benefit most from such methods.
authors.affiliation: Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Goethe University, Frankfurt/Main, Germany.
authors.forename: Solmaz
authors.initials: S
authors.lastname: Sobhanifar
authors.forename: Sina
authors.initials: S
authors.lastname: Reckel
authors.forename: Friederike
authors.initials: F
authors.lastname: Junge
authors.forename: Daniel
authors.initials: D
authors.lastname: Schwarz
authors.forename: Lei
authors.initials: L
authors.lastname: Kai
authors.forename: Mikhail
authors.initials: M
authors.lastname: Karbyshev
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
mesh_terms: D002474:Cell-Free System / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation
chemical_list: D008565:Membrane Proteins
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