title: Development of a multifunctional tool for drug screening against plasmodial protein-protein interactions via surface plasmon resonance.
abstract: We have developed an expression system capable of producing large quantities of low cost, specific peptides that are either His12 -tagged, biotinylated, or unlabeled. The flexibility of this peptide system is suitable for interaction studies via surface plasmon resonance (SPR), co-crystallization, and enzyme-linked immunosorbent assay. Gene blocks containing peptide sequences of interest in addition to a 15 amino acid AviTag™, were cloned into a vector expressing an N-terminal maltose binding protein. The constructs were expressed and purified, and the molecular weights of the recombinant proteins were estimated by analytical size exclusion chromatography. Successful in situ biotinylation of the AviTag was confirmed by anti-biotin western blot and was used for coupling to the surface plasmon resonance chip. We were able to validate, as a proof of concept study, the specific protein-protein interaction of Plasmodium falciparum aldolase (PfAldolase) with three different cytoplasmic adhesin tail peptides from the family of thrombospondin-related anonymous proteins (TRAPs), and to determine their affinities. This method of peptide production enables high yield production of peptides in a two-day, cost effective manner. This tool will allow us to screen for protein-protein interaction inhibitors directed toward the liver stage and blood stage complexes of the glideosome in Plasmodium species. Adaptation of this tool will allow researchers to pursue their own studies of protein-protein interactions.
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins Malaria Research Institute, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Boucher
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bosch
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019921:Biotinylation; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics; D056656:Lab-On-A-Chip Devices; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013194:Staining and Labeling; D020349:Surface Plasmon Resonance
chemical_list: D010455:Peptides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C069428:thrombospondin-related adhesive protein, protozoan; D005634:Fructose-Bisphosphate Aldolase
keywords: AviTag; Plasmodium; crystallography; drug screening; protein-protein interaction; purification; surface plasmon resonance