Cell-free expression and stable isotope labelling strategies for membrane proteins Export

@article{citeulike:5447151, abstract = {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.}, author = {Sobhanifar, Solmaz and Reckel, Sina and Junge, Friederike and Schwarz, Daniel and Kai, Lei and Karbyshev, Mikhail and L\"{o}hr, Frank and Bernhard, Frank and D\"{o}tsch, Volker}, citeulike-article-id = {5447151}, citeulike-linkout-0 = {http://dx.doi.org/10.1007/s10858-009-9364-5}, citeulike-linkout-1 = {http://www.springerlink.com/content/081t7084440555l2}, doi = {10.1007/s10858-009-9364-5}, journal = {Journal of Biomolecular NMR}, keywords = {cell-free, imp-nmr}, posted-at = {2009-10-08 21:42:23}, priority = {2}, title = {Cell-free expression and stable isotope labelling strategies for membrane proteins}, url = {http://dx.doi.org/10.1007/s10858-009-9364-5} }

TY - JOUR ID - citeulike:5447151 L3 - citeulike-article-id:5447151 N2 - 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. JF - Journal of Biomolecular NMR TI - Cell-free expression and stable isotope labelling strategies for membrane proteins KW - cell-free KW - imp-nmr AU - Sobhanifar, Solmaz AU - Reckel, Sina AU - Junge, Friederike AU - Schwarz, Daniel AU - Kai, Lei AU - Karbyshev, Mikhail AU - Löhr, Frank AU - Bernhard, Frank AU - Dötsch, Volker UR - http://dx.doi.org/10.1007/s10858-009-9364-5 ER -