Principal Investigators
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P2 Volker Dötsch
Professor
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Institute of Biophysical Biochemistry,
Biocenter
Goethe-University Frankfurt a.M.
Max-von-Laue-Str. 9
60438 Frankfurt am Main, Germany
Phone +49 (0)69 79 82 96 31
Fax +49 (0)69 79 82 96 32
> send email
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P2 Frank Bernhard
Group Leader
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Institute of Biophysical Biochemistry,
Biocenter
Goethe-University Frankfurt a.M.
Max-von-Laue-Str. 9
60438 Frankfurt am Main, Germany
Phone +49 (0)69 79 82 96 20
Fax +49 (0)69 79 82 96 32
> send email
> homepage
P2 Synergies of cell-free expression systems and NMR spectroscopy for the analysis of membrane proteins
Cell-free expression techniques have emerged in recent times as powerful tools for the fast and efficient production of membrane proteins. Most central problems associated with conventional cellular expression systems are eliminated and the direct synthesis into defined artificial hydrophobic environments like detergents or liposomes enables completely new strategies for membrane protein production (Fig. 1). In addition, reaction protocols of cell-free expression systems can be individualized according to the specific requirements of particular target proteins. This exceptional versatility guarantees high success rates for the production of even complex membrane proteins.
We have established throughput strategies for the optimization of cell-free reaction protocols for the expression of membrane proteins as integrated processes with robotic platforms (Fig. 2). The potentials of cell-free expression for the synthesis and characterization of a selection of electrochemical potential-dependent transporters involved in transport, efflux, signalling, metabolism or biosynthesis will be explored. Individual cell-free expression protocols for medically relevant transporters at high quality will be optimized or developed. The function of selected transporters solubilized in micelles and inserted into liposomes will be characterized in vitro by an array of complementary techniques. Structural approaches based on unique synergic effects of cell-free expression systems in combination with NMR spectroscopy and X-ray crystallography will be explored (Fig. 3). The project will provide a comprehensive pilot study of new applications generated by the cell-free production of membrane proteins.
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- Fig. 1: CF expression modes for high level membrane protein production. Schematic view of cell-free approaches for the production of membrane proteins. P-CF: Membrane proteins are synthesized in the absence of supplied hydrophobic compartments and precipitate after translation. D-CF: Membrane proteins are kept in soluble form by insertion into provided detergent micelles. L-CF: Membrane proteins can become inserted into preformed liposomes, bicelles or nanodiscs of defined composition.
Fig. 2: Throughput design for the optimization of cell-free membrane protein production. An integrated process for pipetting, incubation, separation, purification, quantification and parameter selection of cell-free membrane protein expression conditions has been developed. The expression screening is demonstrated with an example of Mg2+/K+ ion concentration optimization.
Fig. 3: Transmembrane segment (TMS) labelling of membrane proteins by cell-free expression. Specific labelling of amino acid types prevalent in transmembrane regions significantly reduces the complexity of NMR spectra and facilitates the structural analysis of these areas. [15N, 1H] TROSY spectra of A: Uniformly 15N-labelled presenilin-1-CTF and B: TMS-labelled presenilin-1-CTF.
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Publications
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Junge, F., Schneider, B., Reckel, S., Schwarz, D., Dötsch, V. and Bernhard, F. (2008) Large scale production of functional membrane proteins. Cell Mol Life Sci 65, 1729-55.
Reckel, S., Sobhanifar, S., Schneider, B., Junge, F., Schwarz, D., Durst, F., Löhr, F., Güntert, P., Bernhard, F. and Dötsch, V. (2008) Transmembrane segment enhanced labeling as a tool for the backbone assignment of α-helical membrane proteins. Proc Natl Acad Sci USA 105, 8262-7.
Schwarz, D., Junge, F., Durst, F., Frölich, N., Schneider, B., Reckel, S., Sobhanifar, S., Dötsch, V. and Bernhard, F. (2007) Preparative scale expression of membrane proteins in E. coli based continuous exchange cell-free systems. Nat Protocols 2, 2945-57.
Klammt, C., Srivastava, A., Eifler, N., Junge, F., Beyermann, M., Schwarz, D., Michel, H., Dötsch, V. and Bernhard, F. (2007). Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation. FEBS J 274, 3257-69.
Klammt, C., Schwarz, D., Eifler, N., Engel, A., Piehler, J., Haase, W., Hahn, S., Dötsch, V. and Bernhard, F. (2007) Cell-free production of G-protein coupled receptors for functional and structural studies. J Struct Biol 158, 482-93.
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Collaborations
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Glaubitz (P6), Michel (P3), Fendler/Bamberg (P10), Kühlbrandt (P1), Abele (P9), Schwalbe (P13), Tampé (P16), Mäntele (P5), Prisner (P7)