Date of Award
Master of Science in Chemical Sciences (MSCB)
Dr. Jonathan McMurry
Dr. Carol Chrestensen
Dr. Scott Nowak
Over the last decade a number of peptides that are rapidly internalized by mammalian cells have been discovered or designed. Cell-penetrating peptides (CPPs) are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargoes to the cell interior. We have developed a novel CPP-adaptor protein technology that allows any user-defined cargo delivery and release into the cytoplasm. Our hypothesis is that a CPP-adaptor with a moiety allowing high-affinity but reversible non-covalent cargo binding would lead to more efficient penetration and release than current CPP delivery strategies. Delivery of proteins to the interiors of cells has many applications. In addition to detecting and mapping the location of the components of living cells with fluorescent tags in real time, the availability of our system will likely enable the manipulation of signaling pathways and gene expression by allowing the introduction of components, e.g. constitutively active kinases, repressors or enhancers.
CPP-adaptor, TaT-Calmodulin, and cargo proteins (horse radish peroxidase, myoglobin and beta-galactosidase) were expressed and purified from E. coli BL21 (DE3)pLysS. Optical biosensing experiments demonstrated that affinity and kinetics between the novel CPP and cargo proteins did not significantly differ from wild-type interactions; all had subnanomolar affinities. Cargo proteins were labelled with DyLight 550. CPP-cargo complexes or cargo alone were incubated with subconfluent baby hamster kidney, HEK 293T and HT-3 cells. After washing, cells were imaged by fluorescence confocal microscopy. All users define cargos exhibited penetration and release to the cytoplasm whereas cargo-only controls exhibited no measurable penetration (though some adherence to the outside of the cells was observed). Time courses and dose-dependency studies characterizing penetration and release kinetics will be presented as will initial efforts to deliver cargo that may alter cell-signaling pathways.
The results presented herein demonstrate the feasibility of delivering a wide variety of cargo proteins to the intracellular environment; creating an array of potential research, diagnostic and therapeutic applications.
Ngwa, Verra M., "Novel Cell Penetrating Peptides Effect Endosomal Escape and Deliver Protein Cargos into Living Cells" (2016). Master of Science in Chemical Sciences Theses. 10.