Cloning And Expression Of Skp1 And Rav2 From Saccharomyces Cerevisiae In Escherichia Coli

V-ATPases (vacuolar ATPases) are present in the plasma membrane and vacuoles of eukaryotic cells where the V-ATPases function to both acidify intracellular compartments and transport protons across the plasma membrane. V-ATPases could rapidly and reversibly disassemble into free V1 and Vo domains at low extracellular glucose concentrations. RAVE (regulator of the H+-ATPase of the vacuolar and endosomal membranes) plays an important role in regulating the glucose-dependent reversible assembly of the V-ATPase. RAVE is composed of three subunits, Ravlp, Rav2p, and Skplp. There are few reports about the structure and characteristics of RAVE so far.The skpl was cloned from Saccharomyces cerevisiae BY4742 with primers designed according to the sequence of skpl and pET-21c. skpl was digested with Nde I and Not I, and then inserted into corresponding sites of pET-21c vector previously cut with the same restriction enzymes, resulting in the expression plasmid pET-21cSl. The plasmid pET-21cSl was verified by PCR and double enzyme digestion. The nucleotide sequence of the inserted fragment was confirmed by DNA sequencing. Induced with 1 mmol/L IPTG at 25℃and 150 r/min for 4 hours, E. coli BL21(DE3) cells harboring pET-21cSl expressed Skplp. The soluble form of Skplp was purified by Ni-NTA resin.The recombinant plasmid pUCTR2 was constructed for sequencing after rav2 was cloned from Saccharomyces cerevisiae BY4742. Plasmids pUCTR2 and pET-21c were digested with Nde I and Not I, and then the rav2 was ligated with pET-21c resulting in the plasmid pET-21cR2. The plasmid pET-21cR2 was verified by PCR and double enzyme digestion. Induced with 1 mmol/L IPTG at 25℃and 150 r/min for 2 hours, E. coli BL21(DE3) cells harboring pET-21cR2 expressed Rav2p. But in this condition, the expressed target protein formed inclusion bodies. In search of better expression condition, we found that adding of 0.5% glucose into the medium with 0.5 mmol/L IPTG at 16℃and 80 r/min of shaking improved the solubility of Rav2p. The soluble form of Rav2p was purified by Ni-NTA resin.According to the SOPMA analysis, the Rav2p of Saccharomyces cerevisiae has 46.72% of a helix,17.66% ofβfold,4.84% ofβcorner and 30.77% of random coil. BLAST analysis from NCBI revealed that Rav2p sequences shares 49%,49% and 45% identity with ZYRO0C04554p of Zygosaccharomyces rouxii, Kpol₁064p5 of Vanderwaltozyma polyspora and AGL104Cp of Ashbya gossypii, respectively. Analysis of the predicted amino acid sequence revealed that the Rav2p is an unknown function protein of the Rogdi-lz family. Rav2p has 20 sulfur-containing amino acids and 13 prolines, which could be the important reason that Rav2p easily formed inclusion bodies in E. coil. The rav2 and skpl were amplified by PCR using the recombinant plasmid pET-21cR2 and pET-21cSl as templates, respectively. Then rav2 was inserted into pETDuet-1 between the BamH I and Sal I sites. The recombinant plasmid pETDuet-R2 was transformed into BL21(DE3). The Rav2p was expressed after inducing with 1 mmol/L IPTG at 25℃and 150 r/min for 2 hours. Plasmid pETDuet-R2 and skpl were digested with Nde I and Xho I, and then ligated resulting in plasmid pETDuet-R2Sl. E. coli BL21(DE3) cells harboring pETDuet-R2Sl did not express Rav2p and Skplp under the same condition as pETDuet-R2 expression. Skplp was expressed after inducing for 2 h. Skplp and Rav2p were both expressed after inducing for 4 h when the condition changed to 37℃and 150 r/min.