亲和纯化酿酒酵母RAVE复合物

RAVE(Regulator of the H+-ATPase of the Vacuolar and Endosomal membranes) is an essential factor of assembly and reversible disassembly of V-ATPase, and has three subunits, which are Rav1p, Rav2p and Skp1p. It is very important to study structure of RAVE complex to understand more about the regulation of the assembly and reassembly at V-ATPase, but just have few studies about it. In this study, RAVE complex was directly purified from wild strain Saccharomyces cerevisiae BY4742 through FLAG tag that been added to C-terminus of subunit Rav2p. Yeast cells were incubated in 8 L YPD medium at 30 ℃, 200 rpm (OD around of 3). Furthermore harvested cells and broken by a French pressure cell disruptor at 25,000 P.s.i in TBSE (50 mM Tris/Cl, 150 mM NaCl, 1 mM EDTA, pH 7.4) added 1mm PMSF. The cell lysate was centrifuged at 20,000 xg for 20 minutes at 4 ℃. Then, the supernatant loaded onto a small column contained 1 ml of anti-FLAG M2 gel. After washing anti-FLAG column with TBSE, RAVE complex was eluted with TBSE containing 100 μg/ml FLAG peptides. Then, the complex to run SDS-PAGE and identifiy protein bands by immunoblotting and mass spectrometry. The result shows that RAVE complex successfully purified from the strain that the C-terminus of Rav2p with FLAG tag.