brucei

procyclics as previously described (Medina-Acosta & Cross, 1993). Putative genes encoding ME paralogues were identified by blast searching of the T. brucei and T. cruzi genome project database (http://tritrypdb.org/tritrypdb/). see more Four sets of primers were designed to amplify the ORFs corresponding to T. brucei TbME1 and TbME2, and to T. cruzi TcME1 and TcME2, respectively: 1 tbme1 fw 5′-CATATGTTGGGTCGTTCGTTTAAACTTTG-3 All the forward primers contained NdeI restriction sites (underlined), the reverse primers corresponding to TbME1 (Tb11.02.3130) and TbME2 (Tb11.02.3120) contained XhoI restriction sites, and those for TcME1 (Tc00.1047053505183.20) and TcME2 (Tc00.1047053508647.280) contained EcoRI restriction sites (underlined). The coding sequences were amplified using genomic DNA as template, Pfu-Turbo polymerase (Stratagene) and the specific primers designed on the basis of the data available in the genome projects database (http://tritrypdb.org/tritrypdb/). The PCR settings were 5 min at 95 °C and 25 cycles under the following conditions: 95 °C for 45 s, annealing

temperatures of 55 and 58 °C were used for 45 s for T. brucei MEs and T. cruzi MEs, respectively, and extension at 72 °C for 90 s. A final extension step was performed at 72 °C for 10 min. In each of the four reactions, a single fragment (≅1.8 kb) was amplified; upon agarose gel purification the PCR products were cloned into pGEM-Teasy® vector and fully sequenced. Then, T. brucei ME1 (TbME1) and ME2 (TbME2), and find more T. cruzi ME1 (TcME1) and ME2 (TcME2), were subcloned into pET28a+ expression vector (Novagen, Darmstadt, Germany). The 5′-ends of the four genes were similarly extended with a nucleotide sequence encoding a 6 × His-tag and a thrombin cleavage site. The plasmids containing the genes encoding TbME1, TbME2 and TcME2 were used to transform Escherichia coli Rosetta (DE3)pLysS. The

plasmid containing the gene encoding TcME1 was Dolichyl-phosphate-mannose-protein mannosyltransferase transformed in E. coli BL21(DE3) cells harbouring the pGro7 plasmid; the latter, upon induction with arabinose, allows the expression of the GroEL/GroES chaperone system (Takara Bio Inc.). Both E. coli strains were grown in Luria–Bertani medium containing 34 or 20 μg mL1 chloramphenicol, and 30 μg mL1 kanamycin at 37 °C, until an OD600 nm of 0.6 was reached. The expression of T. brucei MEs and T. cruzi ME2 was induced by adding isopropyl-α-d-thiogalactopyranoside (IPTG) to a final concentration of 0.1 and 0.2 mM, respectively. The cultures were further grown for 4 h at 20 °C. In the case of TcME1, the expression was induced by adding IPTG and l-arabinose to a final concentration of 0.2 and 3.33 mM, respectively, and the cultures were further grown for 4 h at 28 °C.