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Development of a multifunctional tool for drug screening against plasmodial protein–protein interactions via surface plasmon resonance
Author(s) -
Boucher Lauren E.,
Bosch Jürgen
Publication year - 2013
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2292
Subject(s) - biotinylation , surface plasmon resonance , flag tag , peptide , biochemistry , streptavidin , biotin , protein tag , chemistry , recombinant dna , protein–protein interaction , target protein , biology , fusion protein , gene , nanotechnology , materials science , nanoparticle
We have developed an expression system capable of producing large quantities of low cost, specific peptides that are either His 12 ‐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. Copyright © 2013 John Wiley & Sons, Ltd.