The efficient delivery of external proteins from the external milieu to the cytosol of mammalian cells has great potential for both scientific investigations and future therapies. However, when assessing the cellular uptake of proteins, it is often difficult to distinguish between proteins that are stuck in the endosomes and those that have escaped into the cytosol. Here, we describe a method employing the prokaryotic enzyme biotin ligase that overcomes this problem and which can be employed for a highly sensitive quantification of cytosolic protein delivery.
Biotin Biotin ligase Cellular internalization Cytosolic protein delivery Streptavidin Western blot Protein engineering
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The research leading to these methods has received support from the EU/EFPIA Innovative Medicines Initiative Joint Undertaking COMPACT grant no 115363. W.P.R.V. was supported by a Rubicon grant (Project number: 825.12.002) from the Netherlands Organisation for Scientific Research and the Forschungskredit from the University of Zurich.
Cronican JJ, Thompson DB, Beier KT et al (2010) Potent delivery of functional proteins into Mammalian cells in vitro and in vivo using a supercharged protein. ACS Chem Biol 5:747–752CrossRefPubMedPubMedCentralGoogle Scholar
Mohammed AF, Abdul-Wahid A, Huang EH et al (2012) The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP-protein cargos to the cytosol of eukaryotic cells. J Control Release 164:58–64CrossRefPubMedGoogle Scholar
Wadia JS, Stan RV, Dowdy SF (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10:310–315CrossRefPubMedGoogle Scholar
Dunn KW, Mayor S, Myers JN et al (1994) Applications of ratio fluorescence microscopy in the study of cell physiology. FASEB J 8:573–582PubMedGoogle Scholar
Richard JP, Melikov K, Vives E et al (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 278:585–590CrossRefPubMedGoogle Scholar
Verdurmen WP, Luginbühl M, Honegger A et al (2015) Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems. J Control Release 200:13–22CrossRefPubMedGoogle Scholar
Barker DF, Campbell AM (1981) The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase. J Mol Biol 146:451–467CrossRefPubMedGoogle Scholar