title: Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification.
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.
authors.forename: Nazhat
authors.initials: N
authors.lastname: Shirzad-Wasei
authors.forename: Jenny
authors.initials: J
authors.lastname: van Oostrum
authors.forename: Petra H M
authors.initials: PH
authors.lastname: Bovee-Geurts
authors.forename: Lisanne J A
authors.initials: LJ
authors.lastname: Kusters
authors.forename: Giel J C G M
authors.initials: GJ
authors.lastname: Bosman
authors.forename: Willem J
authors.initials: WJ
authors.lastname: DeGrip
mesh_terms: D002846:Chromatography, Affinity; D015870:Gene Expression; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D049329:Nanostructures / Q000737:chemistry*; D012243:Rhodopsin / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D012243:Rhodopsin