Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives

Nat Chem. 2021 Jun;13(6):530-539. doi: 10.1038/s41557-021-00661-x. Epub 2021 Apr 15.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell-Penetrating Peptides / chemistry
  • Cell-Penetrating Peptides / metabolism*
  • HeLa Cells
  • Humans
  • Hydrolases / metabolism
  • Immunoglobulin G / metabolism
  • Protein Conformation
  • Protein Transport
  • Proteins / chemistry
  • Proteins / metabolism*
  • Sulfhydryl Compounds / metabolism


  • Cell-Penetrating Peptides
  • Immunoglobulin G
  • Proteins
  • Sulfhydryl Compounds
  • Hydrolases
  • haloalkane dehalogenase