Date of Award
Dr. Tim Hayes
Dr. Lori Hensley
Dr. Wesley Kluck
The E5-TAT-mCherry protein is designed to initiate cellular uptake through endocytosis and react to the acidification of the endosomes by causing membrane lysis and cytosolic delivery. The cell penetrating peptide TAT from the HIV transcription activator binds to the cellular membrane and transports attached or coincubated cargo into the cell through endocytosis. E5 is a derivative of the fusogenic peptide HA2 from the influenza hemagglutinin protein and reacts to the acidification of the endosomal lumen by inserting into the membrane and increasing membrane permeability. The attached red fluorescent protein mCherry serves as a convenient biological probe for cell based assays. Cytosolic delivery has been achieved using E5-TAT-mCherry, although efficiency still remains low. It is thought that imitating the triple stranded coiled coil state of the hemagglutinin protein will lead to increased membrane lysis and greater cytosolic delivery. Therefore, in order to create a more native-like state and to increase the local concentration of the protein within the endosome, a pH-sensitive isoleucine zipper (IZdep) has been incorporated into the sequence. Under acidic conditions, such as those within endosomes, the pH-dependent IZ forms a triple stranded coiled coil. E5-TAT-IZdep-mCherry was express in E. coli and purified with chitin beads and ion exchange chromatography. Native PAGE analysis indicates that two oligomerization states exist for E5-TAT-IZdep-mCherry, however, gel filtration shows only one band. An in vitro erythrocyte lysis assay was used to determine the amount of membrane lysis induced by the protein complex. Very little hemolysis was observed, and surprisingly, microscopic images indicated that the protein did not bind to the inner leaflets of the RBC ghosts as was expected. These results indicate that IZ is not effectively causing the trimerization of the mosaic protein and that the positvely charged TAT peptide is interacting with the negatively charged residues on the IZ sequence which decreases the membrane binding efficiency.
Nickel, Valerie, "The Influence of a Coiled-coil Isoleucine Zipper on the Trimerization and Endosomolytic Activity of the E5-TAT-mCherry Protein" (2013). Honors Theses. 1.