IDENTIFICATION OF PROTEIN-PROTEIN INTERACTIONS AMONG PIF1, PIF2 AND PIF3 OF HEARNPV AND MABRNPV BY YEAST 2 HYBRIDIZATION
Six proteins (HaPIF1, HaPIF2, HaPIF3, MbPIF1, MbPIF2 and MbPIF3) were analyzed by the TMHMM software to predict their membrane topology. Truncated pifs ORFs excluding TM domains were amplified and ligated into pGBKT7 and pGADT7 vectors to produce bait and prey plasmids respectively. The plasmids were transformed into bacteria strain DH10β and the interaction assays were performed in yeast strain AH109 in which the plasmids were finally transformed. It was revealed that HearNPV and MabrNPV PIFs, 1, 2 and 3, interacted with each other both homogenously and heterogeneously. Interaction alone is not enough for functional PIF complex formation but stable co-interaction of PIF complex constituents. Protein interactions between HaPIFs (PIF1, PIF2 and PIF3) and their MbMNPV homologues were observed. The classical yeast two-hybrid analysis remains a reliable technique in determining protein interaction, however functional relevance of interaction in this system continually needs confirmation by more stringent accurate methods emulating their natural physiological environments.