title: Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems.
abstract: Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.
authors.affiliation: Dept of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland. Electronic address: W.Verdurmen@bioc.uzh.ch.
authors.forename: Wouter P R
authors.initials: WP
authors.lastname: Verdurmen
authors.affiliation: Dept of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland. Electronic address: M.Luginbuehl@bioc.uzh.ch.
authors.forename: Manuel
authors.initials: M
authors.lastname: Luginbühl
authors.affiliation: Dept of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland. Electronic address: Honegger@bioc.uzh.ch.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Honegger
authors.affiliation: Dept of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland. Electronic address: Plueckthun@bioc.uzh.ch.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D036002:ADP Ribose Transferases / Q000494:pharmacology*; D017089:Ankyrin Repeat; D001427:Bacterial Toxins / Q000494:pharmacology*; D001692:Biological Transport; D019731:Carbon-Nitrogen Ligases / Q000494:pharmacology*; D002352:Carrier Proteins / Q000494:pharmacology*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000494:pharmacology*; D005098:Exotoxins / Q000494:pharmacology*; D006801:Humans; D061986:MCF-7 Cells; D015202:Protein Engineering; D012097:Repressor Proteins / Q000494:pharmacology*; D037521:Virulence Factors / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D019731:Carbon-Nitrogen Ligases; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D012097:Repressor Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C058125:birA protein, E coli
keywords: Anthrax toxin; Biotin ligase; Cytoplasmic delivery; Designed ankyrin repeat protein; Protein engineering; Pseudomonas exotoxin A