title: A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins.
abstract: Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments - random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR - that enable most portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Hakeenah F
authors.initials: HF
authors.lastname: Brown
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Sudip
authors.initials: S
authors.lastname: Khadka
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: LaCount
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005822:Genetic Vectors; D010957:Plasmids; D010963:Plasmodium falciparum / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins; D003850:Deoxyribonuclease I