Since their first discovery a little over 30 years ago, cell-penetrating peptides have been the subject of intensive research. Nevertheless, there is much less work, and much more controversy, when it comes to the delivery of entire proteins using CPPs. Early reports claim that while small molecules and peptides can be taken up in a non-endocytic manner, protein- CPP conjugates would enter cells exclusively through endocytosis – and often remain trapped in endosomes. In this thesis, the delivery of proteins using cell-penetrating peptides is at the centre-stage. In several different contexts, protein delivery could in fact be achieved in an energy-independent manner. Initially, a pair of GFP-nanobodies were chosen as an easily applicable cargo. The small antibody fragments were engineered to allow modification of at the C-termini using expressed protein ligation. Cyclic CPPs (Tat and R10) were attached, and the conjugates could be purified by affinity chromatography using an immobilized form of the anionic polymer heparin. The conjugates could then enter cells and could be used in downstream intracellular applications. To explore the intracellular fate and targetability of CPP conjugates, the fluorescent protein mCherry was recombinantly expressed as a fusion protein with several peptidic targeting sequences and conjugated to a cyclic R10 CPP through either a cleavable disulfide, or noncleavable thioether linkage. The cytosolic delivery of the protein was successful in either case, though it required elevated concentrations. Interestingly, the non-cleavable conjugate showed accumulation in the nucleoli due to the CPP, while the disulfide-linked conjugate showed clean localization to the target structure or compartment. Localization of the protein to mitochondria after delivery could not be achieved. Finally, it was possible to devise a methodology that allows circumventing the harsh concentration dependence of CPP-mediated transport of large protein cargoes. Through usage of a low μM concentration of both the protein-CPP conjugate along with unbound, free CPP, highly efficient delivery can be achieved. The best CPP additives proved to be thiolreactive peptides, that can label specific loci on the cell membrane through which the protein cargo can transduce. This was effective in several cell lines, as well as with many protein cargoes, amongst others full-length IgG antibodies. In future research, these findings could be used in cell-type specific delivery approaches, by labelling only specific cells with CPPs, and in applications with functional antibodies.
