Expression and purification of Syp-VAMP2 complex into native nanodiscs
Caroline Brown, Snehasish Ghosh, Kallol Gupta
Abstract
This is a protocol for the purification of Syp-VAMP2 complex into native nanodiscs.
Steps
Mammalian expression constructs
Thaw Expi-HEK293 cells and passage 3 times
Using a T2A polycistronic vector containing the VAMP2 and Synaptophysin (Syp) genes with FLAG tag, express VAMP2 and Syp in Expi-HEK293 cells using ExpiFectamine transfection reagent for 48h 0m 0s hours.
Spin down transfected cells and rinse in ice-cold PBS.
Solubilization of VAMP2-Syp into native nanodiscs
Resuspend cells expressing VAMP2-Syp into lysis buffer (50millimolar (mM) , 150millimolar (mM) , 10% volume ) supplemented with protease inhibitor tablets.
Lyse cells using nitrogen cavitation (600 psi, 0h 15m 0s minutes).
Remove debris and nuclei using a 4000 rpm centrifugation spin for 0h 10m 0s minutes.
Ultracentrifuge the clarified lysate at 200,000xg for 1h 0m 0s hour at 4°C to collect membranes.
Resuspend membranes in 1.5% polymer solution (ChloroSMA80) and incubate at 4°C for 2h 0m 0s hours.
Post-solubilization centrifuge the sample at 200,000xg for 1h 0m 0s hour to remove insoluble material.
Native nanodisc enrichment using affinity chromatography
Incubate native nanodiscs containing VAMP2-Syp complex with anti-FLAG resin at 4°C with rotation.
Remove supernatant and wash beads extensively with 50millimolar (mM) , 150millimolar (mM) , 10% volume.
Elute nanodiscs using buffer from Step 11 containing 3XFLAG peptide at 10 ug/ml concentration.
