Reconstituting Membrane Protein Complexes in Nanodiscs

The nanoquick protocol^{40 (link)} was adapted to reconstitute the complex into nanodiscs. Proteins were expressed and the membrane was solubilised as described above but instead of GDN a mixture of LMNG:CHS (10:1) was used at a final concentration of 1%. The solubilised protein was incubated overnight with StrepTactin sepharose beads (Cytiva). The beads were washed in Strep Buffer including 0.1% LMNG:CHS (10:1) and then into SEC buffer including 2% glycerol and 0.1 % LMNG:CHS (10:1). A thousand fold molar excess of POPG was added and incubated for an hour, followed by a 20-fold excess of MSP2N2 (purified as described in^{41 (link)}). After 15 min, activated SM-2 BioBeads (BioRad) were added and the mixture was rotated overnight. The protein complex was eluted in Strep Buffer supplemented with 2.5 mM desthiobiotin (Sigma) and subjected to size-exclusion chromatography as above in SEC buffer without detergent.