An improved fragmentation method of Plasmodium falciparum genes for yeast two-hybrid screening
The ability of exported Plasmodium proteins to remodel the host red blood cell (RBC) is an important virulence factor for malaria pathogenesis. There are over 400 exported Plasmodium proteins and more than half of them are considered hypothetical and uncharacterized. I hypothesize that many of these exported proteins may interact with host proteins. Information is limited regarding these particular protein-protein interactions as well as the function of these exported proteins. The long term goal of this research is to identify interactions between Plasmodium falciparum and host proteins. To accomplish this goal I will utilize the Y2H (yeast two-hybrid) assay, which is a well developed method, to identify protein-protein interactions between Plasmodium and host proteins. However P. falciparum proteins are very difficult to express in yeast. The P. falciparum genome is 80% AT, which renders expression in yeast problematic. P. falciparum genes have AT rich nucleotide sequences that may be targeted for polyadenylation in yeast, which often results in a truncated form of the RNA. In an effort to overcome the issues associated with premature cleavage of full length P. falciparum genes in yeast, I developed an improved fragmentation method that allows P. falciparum genes to be expressed as a series of overlapping fragments that contain shorter AT sequences. For this study I designed 45 fragments of MYB2, which encodes a non-exported P. falciparum protein. Such fragments include sixteen 300 bp (base pair) fragments, fifteen 450 bp fragments and fourteen 600 bp. Eighty-six percent of these fragments were expressed in our Y2H bait vector while 14% lacked expression. After screening the 600 bp fragments against our P. falciparum Y2H library, I identified interactions with PFC0365w and PFL1705w. This defined fragmentation approach allowed us to map the interaction domain of MYB2 with PFC0365w and PFL1705w to a fragment as small as 150 bp. Two different in vivo binding experiments (immunoprecipitations and split luciferase assays) were attempted in effort to confirm the Y2H (Y2H) interactions mentioned previously; however I was not able to make any final conclusions from these trials at the conclusion of this study. Nevertheless, data from this project demonstrates that defined gene fragments designed to minimize the AT stretches within MYB2 were able to yield improved expression in yeast.
LaCount, Purdue University.
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