Enabling the cellular delivery and cytosolic bioavailability of functional proteins constitutes a major challenge for the life sciences. Here we demonstrate that thiol-reactive arginine-rich peptide additives can enhance the cellular uptake of protein–CPP conjugates in a non-endocytic mode, even at low micromolar concentration. We show that such thiol- or HaloTag-reactive additives can result in covalently anchored CPPs on the cell surface, which are highly effective at co-delivering protein cargoes. Taking advantage of the thiol reactivity of our most effective CPP additive, we show that Cys-containing proteins can be readily delivered into the cytosol by simple co-addition of a slight excess of this CPP. Furthermore, we demonstrate the application of our ‘CPP-additive technique’ in the delivery of functional enzymes, nanobodies and full-length immunoglobulin-G antibodies. This new cellular uptake protocol greatly simplifies both the accessibility and efficiency of protein and antibody delivery, with minimal chemical or genetic engineering.
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The UniProt Homo Sapiens database used is available at https://www.uniprot.org/proteomes/UP000005640. All other relevant data are included in the article and its Supplementary Information. The plasmids generated for this study are available from the authors upon request.
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We thank members of the Hackenberger laboratory for comments and discussion and K. Kemnitz-Hassanin and I. Kretzschmar for technical support. We thank D. Schumacher and H. Leonhardt for providing the brentuximab antibody. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP 1623, SFB 1449 and RTG 2473) to C.P.R.H. (HA 4468/9-1, 9-2) and M.C.C. (CA 198/8-1, 8-2), the GIF, the German–Israeli Foundation for Scientific Research and Development, the Einstein Foundation Berlin (Leibniz-Humboldt Professorship), the Boehringer-Ingelheim Foundation (Plus 3 award) to C.P.R.H. and the Fonds der Chemischen Industrie (FCI) to C.P.R.H. and A.F.L.S. (Chemiefonds fellowship).
The technology described in this Article for the delivery of cargoes into cells using cell-surface-reactive peptides is part of a patent application (EP 21159630.9) by A.F.L.S., M.L. and C.P.R.H.
Peer review information Nature Chemistry thanks Roland Brock, Shiroh Futaki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Schneider, A.F.L., Kithil, M., Cardoso, M.C. et al. Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives. Nat. Chem. (2021). https://doi.org/10.1038/s41557-021-00661-x