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.
journal_name
Biol Chemjournal_title
Biological chemistryauthors
Shirzad-Wasei N,van Oostrum J,Bovee-Geurts PH,Kusters LJ,Bosman GJ,DeGrip WJdoi
10.1515/hsz-2015-0100subject
Has Abstractpub_date
2015-08-01 00:00:00pages
903-15issue
8eissn
1431-6730issn
1437-4315pii
/j/bchm.just-accepted/hsz-2015-0100/hsz-2015-0100.journal_volume
396pub_type
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