Purification of active human vacuolar H+-ATPase in native lipid-containing nanodiscs

Abstract

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Molecular basis of mEAK7-mediated human V-ATPase regulation

“…Recently, a study showed that intact active V-ATPase could be purified from a HEK293 cell line that stably expressed subunit a4 23 . We took advantage of this remarkable work to seek for modulators under different cell culture conditions.…”

“…The V-ATPase was purified with the nanodiscs scaffold protein MSP1E3D1 and reconstituted into native lipid-containing nanodiscs according to an established protocol (Supplementary Fig. 1a ) 23 .…”

“…The ATP6V0a4 stable cell line was obtained using a similar method as a previous protocol 23 . Briefly, Expi293F cells (Thermo Fisher Cat #A14527) were plated in 10 cm dishes and maintained in high glucose DMEM (Gibco) with 10% FBS and 1% PS at 37 °C with 5% CO 2 .…”