| Assay Method Information | |
| | PI(4)K enzymatic Assay |
| Description: | Baculovirus expression and purification of C. parvum phosphatidylinositol 4-kinase: The full-length coding sequence of C. parvum PI(4)K (cgd8_4500, 1114 amino acids) was codon-optimized for baculovirus expression, synthesized and cloned into pFastBac-HTb (Invitrogen 10584-027) in frame with the amino-terminal polyhistidine tag using the BamHI and HindIII restriction sites. Recombinant pFastBacHTb-CpPI(4)K bacmid clones were generated by site-specific transposition in E. coli DH10Bac (Invitrogen 10361-012). The bacmid sequence was confirmed by direct DNA sequencing to confirm a lack of mutations across the whole gene. The subsequent steps for bacmid isolation, transfection and selection of the recombinant viruses were performed according to the manufacturer s protocol (Bac-to-Bac system # 10359, Invitrogen).SF9 cells, cultured in SF-900 III serum-free medium, were transfected with recombinant baculovirus at 1/200 (v/v) and incubated at 27^°C for 72^h. The pellets were collected after centrifugation and re-suspended in cell lysis buffer (20^mM Tris-HCl, pH^7.5, 300^mM NaCl, 1mM DTT, 20mM imidazole, 0.01% Triton X-100 and 1×^complete protease inhibitor cocktail without EDTA (Roche Diagnostics 04693116001)). The cell suspension was lysed by sonication and the clarified supernatant was loaded onto a 1^ml HisTrap affinity column (GE Healthcare) pre-equilibrated with buffer A (20^mM Tris-HCl, pH^7.5, 300^mM NaCl, 1mM DTT, 20mM Imidazole, and 1×^complete protease inhibitor cocktail without EDTA). The column was washed with buffer B (buffer A containing 45 mM imidazole) and the bound protein of interest was eluted with buffer C (buffer A with 90^mM imidazole). The fractions containing CpPI(4)K were pooled, concentrated using Amicon Ultra-15 and purified by a gel-filtration column (Hi-Load 26/60 Superdex 200, GE Healthcare) equilibrated with 20^mM Tris, pH^7.5, 300^mM NaCl, 1^mM DTT and 1×^protease inhibitor cocktail without EDTA. The concentrations of the purified protein (Mw 132.39 kda) was determined by using the protein molar extinction coefficient (ε280^nm = 133,810^M−1^cm−1). Aliquots were flash frozen in liquid nitrogen and immediately stored at −80^°C.PI(4)K enzymatic Assay: The CpPI(4)K enzymatic assay was performed as described earlier with a some modifications (McNamara et al., 2013). Briefly, L-α-phosphatidylinositol (Avanti Polar Lipid 840046), dissolved in 3% n-octylglucoside (Roche Diagnostics 10634425001), was used as the lipid substrate for the PI(4)K activity assay. CpPI(4)K was assayed using Transcreener ADP2 FP detection kit (BellBrook 3010) in a black, solid 384-well plate (Corning 3575). The final assay volume was 10^μl and contained 3^nM of the respective CpPI(4)K construct in 10^mM Tris, pH^7.5, 1^mM DTT, 3^μM ATP, 5^mM Mn2+, 0.05% Triton X-100 and 10^μM phosphatidylinositol/octylglucoside. The enzyme reaction was performed for 50 minutes at room temperature and was stopped by adding 10^μl of detection mix containing 1×^stop buffer (50mM HEPES, pH7.5, 400mM NaCl, 20mM EDTA, and 0.02% Brij-35), 2^nM AMP Alexa Fluor 633 tracer, and 20^μg^ml−1 ADP antibody. Fluorescence polarization measurements were performed on the Infinite M1000 plate reader (Tecan) with λex = 635^nm and λem = 680^nm (20-nm bandwidth). IC50 values were calculated using Graphpad Prism software. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |