Abstract
Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.
Acknowledgments
We thank Dr. Thomas Sakmar (The Rockefeller University, New York, NY, USA) for providing the plasmid for ZAP1-expression. We acknowledge Dr. Rosalie Crouch (Medical University of South Carolina, Charleston, SC, USA) for a generous gift of 11-cis retinal through financial support from NEI. We thank Dr. Vincent Lemieux (Radboud University, Nijmegen, The Netherlands) for support with the DLS assay. This research was supported by funds to WJDG from the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW project 700.54.008).
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