1 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU2 Faculty of Science, SDU3 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU
Nanodiscs are self-assembled soluble discoidal phospholipid bilayers encirculated by an amphipatic protein that together provide a functional stabilized membrane disc for the incorporation of membrane-bound and membrane associated molecules. Integral membrane proteins in nanodiscs adopt a uniform environment that resembles biological membranes and where the lipid composition in the immediate surroundings of the protein can be controlled. Additionally, its oligomerization state can be varied by careful control of protein:MPS stoichometry and by selection of MSP with a suitable length that leads to a disc area that can accommodate the desired oligomerization state. The scope of the present work is to investigate how Nanodiscs can be employed for affinity purification of interaction partners from complex mixtures. Cholera Toxin and its glycolipid receptor GM1 constitute a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodiscs that contain the glycolipid receptor GM1 in lipid bilayers, enabling facile elution from solid supports. By a combining surface plasmon resonance (SPR, Biacore) measurements and micro-scale affinity chromatography we have optimized the immobilization, binding, washing, and elution conditions for affinity chromatography. Finally, we demonstrate that GM1 in nanodiscs can be used for co-immunoprecipitation with FLAG antibody-coated agarose beads from enterotoxigenic E. coli lysates. The co-immunoprecipitated protein was identified by mass spectrometry as heat-labile toxin, a homolog of cholera toxin.