Nickel nitrilotriacetic acid agarose resin

Full-length TcPI3K gene was amplified using the following primers carrying hemi-restriction sites: PI3K-pET-Fw-BamHI 5 -GGATCCGTCTTCCCCCAGCGAATTGTT-3 and PI3K-pET-Rv-HindIII 5 -AAGCTTATGTGTCATAATATGAATATAGTCA-3 , cloned into pGEM-T Easy plasmid (Promega, Madison, WI). To express the N-terminal His-tagged TcPI3K in Escherichia coli, the coding sequence was transferred from pGEM-T Easy into the pET-22b(+) expression vector (Invitrogen). The construct [pET-22b(+)-TcPI3K] was transformed into BL21(DE3) pLysS host (E. coli B, F -, dem, ompT, hsdS, (rb -, mB -), gal -(DE3), [pLysS, camr]), and the recombinant protein was induced with 300-500 M isopropyl-1-thio-d-galactopyranoside (IPTG) at 25 • C, 30 • C or 37 • C for 3-16 h. Cells were harvested by centrifugation and resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100) containing a protease inhibitor mixture. The protein extract was prepared by sonication and centrifuged at 10,000 × g; the obtained pellet (P10 fraction) and supernatant (S10 fraction) were stored at -20 • C. The S10 fraction was used for purification using a nickel-nitrilotriacetic acid-agarose resin (Invitrogen) and eluted with lysis buffer containing 20-200 mM imidazole.