Direct protein delivery to mammalian cells using cell-permeable Cys2-His2 zinc-finger domains

J Vis Exp. 2015 Mar 25:(97):52814. doi: 10.3791/52814.

Abstract

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cell Membrane Permeability
  • Cysteine / chemistry*
  • DNA-Binding Proteins / administration & dosage
  • DNA-Binding Proteins / chemistry
  • HeLa Cells
  • Histidine / chemistry*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Proteins / administration & dosage*
  • Proteins / chemistry
  • Zinc Fingers*

Substances

  • DNA-Binding Proteins
  • Proteins
  • Histidine
  • Cysteine