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Incorporation of membrane proteins into lipid bilayers


Nanodisc with cell-free expressed bacteriorhodopsin (bR, purple), an α-helical transmembrane retinal protein (originally from H.salinarum). Immobilized by His-Tag to Ni-NTA self-assembled monolayer (SAM)

Cell-free expression systems in combination with nanodiscs (discoidal lipid bilayer particles) offer a valuable approach to probe membrane proteins in situ. To express a protein by a cell-free system, purified and separated components from cell lysates, e.g. of prokaryotic or eucaryotic origin, get re-combined with certain additives (e.g. amino acids, energy or buffer components, transcription enzymes) in a test tube in a respective manner to process a complete bio-protein synthetic cycle of an offered gene, which encodes for a certain protein. Customization of the system and offering of a lipid support (here: nanodiscs) enable membrane protein expression in a high yield.
Due to the nature of nanodiscs common tagging methods (e.g. His-Tag, Biotin-Tag) can be applied to immobilize the lipid support to various substrates (e.g. to Au-SAM modified by Ni-NTA or passivated glass substrates offering streptavidin entities).
Currently we focus on membrane protein folding in situ using cell-free expression systems and nanodiscs to monitor synthesis and folding events of bacteriorhodopsin during the protein translation by the ribosome. (A. Baumann et al., 2014 manuscript in preparation)

Contact: Dr. Iris v. d. Hocht