13 000 rpm for 30 min at 4 C. Protein concentra tions in the supernatants were determined using the BCA protein assay. Recombinant proteins e pression and purification Initial e periments with the wild type PfI2 Sunitinib supplier cDNA did not allow the production of recombinant protein whatever the bacterial plasmid and the condition of e pression used. In order to overcome this problem, a PfI2 gene with opti mized codons has been synthesized. The sequence is presented in Additional file 5 Figure S2. This synthetic gene has been cloned in different bacterial and yeast plasmids for interaction and functional studies and used as template to obtain deleted and mu tated PfI2 proteins. Briefly, the full length coding region of PfI2WT, PfI2 and PfI2 were obtained by PCR with the primers Pr1 Pr2, Pr3 Pr4 and Pr5 Pr6 re spectively and subcloned in pQE30.

For the e pression of PfPP1, the pETDuet 1 e pression system was used. The re striction sites are mentioned in Additional file 1 Table S1. Before cloning in e pression vectors, Inhibitors,Modulators,Libraries all PCR products were subcloned in a pCR2. 1 TOPO vector and verified by sequencing for the absence of any modifi cation introduced by Taq polymerase. To obtain the PfI2 mutant constructs, we performed PCR based site directed mutagenesis using the construc tions pQE30 PfI2 or pGADT7 PfI2 as templates, the primers Pr7 Pr8 or Pr9 Pr10 and using Isis Proofreading DNA polymerase. The PCR conditions consisted of 1 min at 95 C followed by 16 cycles at 95 C, 55 C and 72 C. The parental DNA plasmid was then digested with DpnI and an aliquot was used to transform L10 Gold Ultracompetent cells.

Mutated plasmids were checked by se quencing for the replacement of tryptophan 16 and tyrosine 103 by an alanine and then used for the e pression of mu tant PfI2 recombinant proteins Inhibitors,Modulators,Libraries or yeast two hybrid assays. Protein e pression was carried out in the E. coli M15 strain for the pQE30 construct Inhibitors,Modulators,Libraries and the BL21 strain for pETDuet 1 constructs. The e pression of His6 PfI2 pro teins was carried out in the presence Inhibitors,Modulators,Libraries of 0. 5 mM IPTG at 37 C for 2 hr. For the e pression of His6 PfPP1, the culture was induced overnight at 16 C in the presence of 0. 5 mM IPTG and 1 mM MnCl2. Cells were harvested in sonication buffer. His tagged recombinant proteins were purified according to manufacturers instructions by Ni2 chelation chroma tography.

With respect to the His6 PfI2 proteins, the e tract was prepared using a 20 mM Tris HCl, 150 mM Nacl, 20 mM Imidazole and 6 M guanidine buffer and loaded on a 1 ml nickel NTA resin column. Washing steps were performed with a buffer containing 20 mM Tris HCl, 150 mM NaCl and 20 mM imidazole. The imidazole eluted proteins Brefeldin_A were dialyzed against 20 mM Tris pH 7. 4, 150 selleck chemicals Erlotinib mM NaCl. Under these conditions, the purity checked by SDS PAGE followed by Coomassie blue staining was 95%. His6 PfI2 protein was further sub jected to peptide mass fingerprint by MALDI TOF mass spectrometry to confirm its identity. For antisera production, the purified His6 PfI2WT was