Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems

J Control Release. 2015 Feb 28;200:13-22. doi: 10.1016/j.jconrel.2014.12.019. Epub 2014 Dec 17.

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.

Keywords: Anthrax toxin; Biotin ligase; Cytoplasmic delivery; Designed ankyrin repeat protein; Protein engineering; Pseudomonas exotoxin A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP Ribose Transferases / pharmacology*
  • Ankyrin Repeat*
  • Bacterial Toxins / pharmacology*
  • Biological Transport
  • Carbon-Nitrogen Ligases / pharmacology*
  • Carrier Proteins / pharmacology*
  • Cell Line
  • Cytoplasm / metabolism
  • Escherichia coli Proteins / pharmacology*
  • Exotoxins / pharmacology*
  • Humans
  • MCF-7 Cells
  • Protein Engineering
  • Repressor Proteins / pharmacology*
  • Virulence Factors / pharmacology*

Substances

  • Bacterial Toxins
  • Carrier Proteins
  • Escherichia coli Proteins
  • Exotoxins
  • Repressor Proteins
  • Virulence Factors
  • ADP Ribose Transferases
  • toxA protein, Pseudomonas aeruginosa
  • Carbon-Nitrogen Ligases
  • birA protein, E coli