Preparation of Constructs Containing Human Receptor Sequences and PfEMPl Binding Domains and an Investigation of their Compatibility with the UALmembrane® Yeast Two-Hybrid System.
Winkler, Laura L.
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Malaria kills more than a million people each year. Although there are several strains of Plasmodium parasite that cause malarial infections in humans, infections caused by Plasmodiumfalciparum are characterized asthe most common and deadly in humans. Pathogenesis from Plasmodiumfalciparum infection occurswhen these organisms infiltrate red blood cells and transcribe a certain set of malarial proteins, termed Plasmodiumfalciparum Erythrocyte Membrane Protein-Is (PfEMPls). These proteins are inserted into the membranes of infected red blood cells and are implicated in the adherence and sequestration of infected red blood cells to human receptors throughout the body. In this study, constructs were created and initially tested for compatibility in the DUALmembrane® yeast two-hybrid system, an assay that may be used to identify membrane protein interactions between human receptors and PfEMPl binding domains that are associated with mild malaria. To generate the constructs for the DUALmembrane® yeast two-hybrid system, two different cloning methods were utilized and compared. Constructs containing CD31 and VCAM-1 human receptor sequences were created using a TOPO® cloning method, whereas the classical cloning technique proved unsuccessful. Plasmids containing PfEMPl binding domains, specifically PF08_0107 and PFD0995c domains, were also generated using TOPO® cloning. Transfection of NMY51 yeast cells with the prepared constructs resulted in very few positive colonies, indicating poor transfection efficiencies or an inability to fully lyse the cells during colony PCRverification. Western analysis revealed a lack of protein expression in CD31 and PF08_0107 positive colonies, suggesting that this particular system does not properly express the prepared constructs or that protein expression was below levels of detection. Further experimentation is needed to determine whether these constructs are indeed compatible with the DUALmembrane® yeast two-hybrid system.