Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification
SourceBiological Chemistry, 396, 8, (2015), pp. 903-15
Article / Letter to editor
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SubjectRadboudumc 0: Other Research RIMLS: Radboud Institute for Molecular Life Sciences; Radboudumc 12: Sensory disorders RIMLS: Radboud Institute for Molecular Life Sciences; Radboudumc 19: Nanomedicine RIMLS: Radboud Institute for Molecular Life Sciences
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.
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