Cell-free expression and stable isotope labelling strategies for membrane proteins |
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Authors: | Solmaz Sobhanifar Sina Reckel Friederike Junge Daniel Schwarz Lei Kai Mikhail Karbyshev Frank L?hr Frank Bernhard and Volker D?tsch |
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Institution: | (1) Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe University, Frankfurt/Main, Germany;(2) Cluster of Excellence Frankfurt (Macromolecular Complexes), Goethe University, Frankfurt/Main, Germany; |
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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 α-helical transmembrane proteins which benefit most from such methods. |
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