University of Kentucky

Poster Title

Application of Phage Display Technology to Establish Binding Partners to the Autophagy Protein ATG8 from Evolutionarily Diverse Organisms

Institution

University of Kentucky

Abstract

The parasite, Toxoplasma gondii (Tg) is capable of infecting virtually all warm-blooded animals and is estimated to have infected one-third of the world's population. Despite considerable progress, there is still much to be learned about this pervasive parasite's infectious mechanism and many other diseases similar, such as malaria. Work in the Sinai laboratory has established the role of autophagy as a death mechanism. The protein ATG8 is a key player in this process. We are working with the autophagy related protein TgATG8 to test phage display technology that can be employed to identify interacting proteins. We are using the protein TgATG8 because we know the potential binding target sequences to be pulled out should be ATG3, ATG4, and ATG7-providing confidence in the identification of new interactions. We have cloned the ATG8 homologs from Toxoplasma, yeast and humans and expressed them in E. coli and His-tagged proteins. These proteins are being used as bait to isolate bacteriophage from a phage display library to identify sequences that bind to all the ATG8 homologs as well as those specific for each other species. The initial screen using TgATG8 as the bait has been completed with the phage plaque assays pointing to significant enrichment of binding sequences. By comparing the binding partners for TgATG8 with those for the yeast and human proteins we expect to gain new insights into this critical factor. In addition we hope to establish a technology platform based on phage display to apply to other proteins from infectious agents.

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Application of Phage Display Technology to Establish Binding Partners to the Autophagy Protein ATG8 from Evolutionarily Diverse Organisms

The parasite, Toxoplasma gondii (Tg) is capable of infecting virtually all warm-blooded animals and is estimated to have infected one-third of the world's population. Despite considerable progress, there is still much to be learned about this pervasive parasite's infectious mechanism and many other diseases similar, such as malaria. Work in the Sinai laboratory has established the role of autophagy as a death mechanism. The protein ATG8 is a key player in this process. We are working with the autophagy related protein TgATG8 to test phage display technology that can be employed to identify interacting proteins. We are using the protein TgATG8 because we know the potential binding target sequences to be pulled out should be ATG3, ATG4, and ATG7-providing confidence in the identification of new interactions. We have cloned the ATG8 homologs from Toxoplasma, yeast and humans and expressed them in E. coli and His-tagged proteins. These proteins are being used as bait to isolate bacteriophage from a phage display library to identify sequences that bind to all the ATG8 homologs as well as those specific for each other species. The initial screen using TgATG8 as the bait has been completed with the phage plaque assays pointing to significant enrichment of binding sequences. By comparing the binding partners for TgATG8 with those for the yeast and human proteins we expect to gain new insights into this critical factor. In addition we hope to establish a technology platform based on phage display to apply to other proteins from infectious agents.