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[…] such a way regulate the parasite's ability to import nutrient (; ) and recycle receptors., Saccharomyces cerevisiae was used as the source organism to study PfRab interactors, as its Ypt protein and effectors are characterized and also because it shares the same number of Rab proteins as Plasmodium falciparum (). The yeast Ypt/Rab genome sequences as well as the physical and genetic interactors were downloaded from SGD database (http://www.yeastgenome.org). Eight hundred twelve interactions involving 596 proteins were found to be involved in the S. cerevisiae Ypt/Rab-interactome network. All the redundant interactions were removed and the remaining interactions represented as networks using Cytoscape (http://www.cytoscape.org/)., The orthologous protein information was obtained by performing pairwise alignment of the sequences using BLASTp at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Orthologue groups were obtained from OrthoMCL DB (http://www.orthomcl.org/cgi-bin/OrthoMclWeb.cgi). BLASTp (E-value‚ȧ0.05) was performed by taking S. cerevisiae protein sequences as queries against P. falciparum protein sequences obtained from PlasmoDB (http://plasmodb.org/plasmo) and confirming the hit by reverse BLAST. The first hit obtained was taken as query for the reverse BLAST against non-redundant (nr) protein database with S. cerevisiae as organism. Homologues were obtained for 200 proteins., We used BLASTp searches to identify the homologues of S. cerevisiae Ypt proteins and their effectors in P. falciparum. For those proteins where reverse BLASTp failed to identify a clear homologue due to the best hit being already assigned, the second best hit was taken as the homologue. Due to a high degree of similarity between Ypt10 and Ypt11, multiple sequence alignments (ClustalW) (http://www.ebi.ac.uk/Tools/clustalw2/index.html) were applied together with PfRab11B and PfRab18., To make C-terminal PfRab7-His, PfRab5A-His, PfRab5B-His and PfRab5C-His constructs for expression in Escherichia coli, the CDS of Pfrab7 (PFI0155c), Pfrab5a (PFB0500c), Pfrab5b (MAL13P1.51) and Pfrab5c (PFA0335w) were PCR amplified from cDNA of P. falciparum (3D7) and cloned into BamHI/XhoI site of the E. coli expression vector pET-21d (Novagen, EMD4Biosciences). The sequences of all expression constructs were verified. To make the N-terminal GST-PfPKA-C construct, a codon optimized synthetic Pfpka-c gene (PFI1685w) was custom made (Eurobio) in order to improve recombinant protein levels when expressed in E. coli., All c […]

Pipeline specifications

Software tools Cytoscape, BLASTP, Clustal W
Databases PlasmoDB
Organisms Saccharomyces cerevisiae, Plasmodium falciparum, Homo sapiens
Diseases Malaria
Chemicals Nucleotides, Cyclic, Ribonucleotides, Heterocyclic Compounds, 2-Ring, Purine Nucleotides