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Novel Cell‐Penetrating Peptides Overcome Endosomal Escape and Deliver Protein Cargos into the Cell
Author(s) -
NGWA Verra,
Salerno John,
Nowak Scott,
Chrestensen Carol,
McMurry Jonathan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.631.2
Subject(s) - cytoplasm , microbiology and biotechnology , endosome , chemistry , signal transducing adaptor protein , endocytosis , cell , green fluorescent protein , biophysics , biochemistry , biology , signal transduction , gene
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 enable the manipulation of signaling pathways and gene expression by allowing the introduction of components, e.g. constitutively active kinases, repressors or enhancers. CPP‐adaptors and cargo proteins (horse radish peroxidase, myoglobin and Beta‐galactosidase) were expressed in 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 (BHK) cells. After washing, cells were imaged by fluorescence confocal microscopy. All user 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 well as initial efforts to deliver cargo that will alter cell‐signaling pathways will be described. The present results demonstrate the feasibility of delivery of a wide variety of cargo proteins and open up an array of potential research, diagnostic and therapeutic applications. Support or Funding Information NIH grant R25GM111565