The supernatant CC5013 was discarded, and the pellet of parasites was stored in liquid N2 for subse quent analysis. and the amplicon cloned in pGEM T easy. After sequencing, the fragment containing the complete ORF was subcloned in Inhibitors,Modulators,Libraries pGEX2T for recombinant protein expression using the introduced BamHI and EcoRI sites. Expression and Purification of rPfFPPS from Escherichia coli Recombinant pGEX 2 T FPPS expression vector was used to transform Escherichia coli BL21 pLys RIL cells. Bacterial clones were grown in LB medium containing 50 ug ml ampicillin and 34 ug ml chloram phenicol at 37 C in Inhibitors,Modulators,Libraries Luria Broth until an OD600 of 0. 6. At this time point, the expression of rPfFPPS was in duced with 0. 2 uM isopropyl B D thiogalactoside at 24 C overnight. Cells were pelleted by centrifugation and resuspended in lysis buffer PBS 0.
1% Triton X 100 pH 7. 2, 0. 05 mg ml lysozyme and 0. 2 mM PMSF. Lysis was completed by sonication. Recombinant proteins were then puri fied using glutathione sepharose beads, following the manufacturers instructions. Proteins were checked for purity by SDS PAGE and quantified by the Bradford method. Enzymatic activity assay Inhibitors,Modulators,Libraries The catalytic activity of rPfFPPS was assayed by measur ing the conversion of IPP to products, by two different protocols, Protocol I The method described by Ling et al. was used with some modifications. Briefly, the assay mixtures contained 10 mM HEPES buffer pH 7. 4, 2 mM MgCl2, 2 mM dithiothreitol, 100 uM IPP, an allylic substrate, and 500 1,000 ng of re combinant protein in a total volume of 100 ul.
The reac tion was carried Inhibitors,Modulators,Libraries out at 37 C for 30 min and stopped by addition of 10 ul of 6 M HCl. The reaction mixture was neutralized by addition of 15 ul of 6 M NaOH. The alco holic products were Inhibitors,Modulators,Libraries then extracted twice with 500 ul hexane and analysed by reverse phase thin layer chroma tography. All non radioactive substrates and chemicals were from Sigma Aldrich. Protocol II rPfFPPS activity was measured by a modification of the method described by Chang et al. Final assay con centrations were 50 mM Tris HCl buffer pH 7. 5, 2 mM MgCl2, 5 mM iodoacetamide, and 500 1,000 ng of re combinant protein. The concentrations of allylic substrate, DMAPP, GPP and FPP were the same as described above. The final reaction volume was 100 ul. After pre incubation at 37 C for 10 min, the reaction was started by adding 50 uM IPP. The mixture was incubated at 37 C for 30 min and the reaction was terminated by addition of distilled H2O and NaCl saturated water. The diphosphate products were then extracted Ganetespib OSA twice with 500 ul of 1 butanol saturated with NaCl saturated water and analysed by reverse phase high performance liquid chro matography. Enzyme activity measurements using FPP and IPP as substrates were also carried out.