Cloning, Expression And Purification Of The Rav1p, A Subunit Of The RAVE Complex From Saccharomyces Cerevisiae

In eukaryotic cells, the V-ATPase is found throughout the endomembrane, includingvesicles, lysosomes, endosomes, synaptic vesicles and Golgi apparatus. As an ATP-drivenproton pump, it acidifies intracellular compartments to maintain the specific pH environmentrequired for many biochemical reactions. The RAVE complex can regulate the activity of theV-ATPase via regulating the reversible dissociation of the V1and V0domains of V-ATPase;however, the mechanism of the V-ATPase regulation is not clear.The RAVE complex is composed of three subunits: Rav1p (155kDa), Rav2p (40kDa)and Skp1p (22.3kDa). The rav1and the vam5were amplified from Saccharomyces cerevisiaegenomic DNA. The rav1was ligated to pACYCDuet-1and pET28a (+) vector, respectively,and the vam5was ligated to pET-21a (+). The recombinant plasmids were named aspACYCDuet-rav1, pET28a-rav1and pET21a-vma5, respectively.The recombinant plasmids were transferred to BL21(DE3) competent cells. Afteroptimization of the expression condition, we found that most soluble Rav1p was obtained if E.coli BL21(DE3) pET28-rav1was grown at37°C to OD600=0.6before adding ofisopropyl-β-D–thiogalactopyranoside (IPTG) to a final concentration of1mmol/L andsubsequently induced at16°C for30h before harvest; most soluble Vma5p was obtained if E.coli BL21(DE3) pET21-vma5was grown at37°C to OD600=0.6before adding of IPTG to afinal concentration of1mmol/L and cultured for another5h before harvest.We purified fusion proteins using the1mL His-Trap column. The results of SDS-PAGEand western blot showed that, the fusion protein Rav1p with both N-and C-terminal His-Tagscan bind to the column efficiently and be eluted by the solution of500mmol/L imidazole; thefusion protein Vma5p with C-terminal His-Tag can be eluted by the solution of300mmol/Limidazole.SWISS-MODEL results showed that N-terminus of Rav1p forms a β-propeller domain.This research predicts that the β-propeller domain of Rav1p can interact reversibly withsubunits E, G and C of the V-ATPase, to regulate the assembly of V-ATPases and the activityof V-ATPases effectively.