Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

Biological Chemistry
Nazhat Shirzad-WaseiW J DeGrip

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 thei...Continue Reading

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Citations

Feb 7, 2016·Progress in Retinal and Eye Research·Nazhat Shirzad-Wasei, Willem J DeGrip
Nov 20, 2015·Chemical Reviews·Yingfeng TuDaniela A Wilson
Feb 9, 2017·Chemical Reviews·Ilia G Denisov, Stephen G Sligar
Dec 8, 2015·European Biophysics Journal : EBJ·Jonas M DörrJ Antoinette Killian
Aug 31, 2016·Science Advances·Hyunwook LeeSusan Hafenstein
Nov 2, 2019·Biochimica Et Biophysica Acta. Biomembranes·Srividya GanapathyWillem J de Grip
Sep 21, 2017·Analytical Chemistry·Tao GaoGenxi Li

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