Cloning and
SGC-CBP30 of UGPPase. A full-length cDNA encoding UGPPase was amplified by PCR from mouse skeletal muscle cDNA (Biochain) using primers specific for the gene encoding the mouse UGPPase, UGPP (NCBI Accession No. NM_025399): 5′-GCTCGGATCCCATATGGAGCGCATCGACGGGGTG-3′ (forward) and 5′-TGGAATTCGGCCTCTCGAGCCCCAAGGTCTCACTG-3′ (reverse). The cDNA was subsequently cloned into a TA vector (Invitrogen), creating pUGPP, and introduced into E. coli DH5-a competent cells. For protein expression, the Nde1/Xho1 fragment of pUGPP was subcloned into the pET28a expression vector (Novagen). E. coli BL21-DE3 transformed with the pET-UGPP plasmid were grown at 37 °C in 500 ml 2× YT medium containing 30 μg/ml kanamycin until an A600 reading of 0.4–0.6 was reached. The culture was induced with 1 mM isopropyl β-d-thiogalactoside, grown 2 h, harvested by centrifugation and resuspended in 10 ml of lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidizole). Cells were lysed by incubating 30 min on ice with 0.5 mg/ml lysozyme followed by sonication. After centrifugation at 20,000g at 4 °C for 30 min, the supernatant was applied to a column containing 2 ml of 50% Ni–NTA resin (Qiagen), washed with 35 mM imidizole buffer, eluted with 250 mM imidizole buffer and dialyzed into 1 L 50 mM Tris–HCl, pH 8.0, 1 mM dithiothreitol. Aliquots were stored at ?80 °C with 46% (v/v)
ethylene glycol.
