Advertisement

Cytotechnology

, Volume 56, Issue 1, pp 41–48 | Cite as

A practical approach for intracellular protein delivery

  • Claire O. Weill
  • Stéphanie Biri
  • Abdennaji Adib
  • Patrick Erbacher
Method in Cell Science

Abstract

Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery.

Keywords

Intracellular release Live cells Non-viral technology Protein delivery Synthetic delivery reagent 

Abbreviations

BSA

Bovine serum albumin

CPP

Cell-penetrating peptide

DLS

Dynamic light scattering

FACS

Fluorescence-assisted cell sorting

PTD

Protein transduction domain

R-PE

R-phycoerythrin

Notes

Acknowledgments

We are grateful to Drs C. Muller and J. Peluso for their help in FACS analysis and to Drs J.-P. Behr and J.-F. Williamson for critical reading of the manuscript.

References

  1. Bar-Sagi D, Feramisco JR (1985) Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell 42:841–848CrossRefGoogle Scholar
  2. Campbell PL, McCluskey J, Yeo JP, Toh BH (1995) Electroporation of antibodies into mammalian cells. Methods Mol Biol 48:83–92Google Scholar
  3. Cassinelli G, Lanzi C, Petrangolini G, Tortoreto M, Pratesi G, Cuccuru G, Laccabue D, Supino R, Belluco S, Favini E, Poletti A, Zunino F (2006) Inhibition of c-Met and prevention of spontaneous metastatic spreading by the 2-indolinone RPI-1. Mol Cancer Ther 5:2388–2397CrossRefGoogle Scholar
  4. Chuck AS, Clarke MF, Palsson BO (1996) Retroviral infection is limited by Brownian motion. Hum Gene Ther 7:1527–1534CrossRefGoogle Scholar
  5. Dalkara D, Zuber G, Behr JP (2004) Intracytoplasmic delivery of anionic proteins. Mol Ther 9:964–969CrossRefGoogle Scholar
  6. Drin G, Cottin S, Blanc E, Rees AR, Temsamani J (2003) Studies on the internalization mechanism of cationic cell-penetrating peptides. J Biol Chem 278:31192–31201CrossRefGoogle Scholar
  7. Erbacher P, Zou S, Bettinger T, Steffan AM, Remy JS (1998) Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. Pharm Res 15:1332–1339CrossRefGoogle Scholar
  8. Farhood H, Bottega R, Epand RM, Huang L (1992) Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity. Biochim Biophys Acta 1111:239–246CrossRefGoogle Scholar
  9. Fischer R, Kohler K, Fotin-Mleczek M, Brock R (2004) A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. J Biol Chem 279:12625–12635CrossRefGoogle Scholar
  10. Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF (2001) Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res 61:474–477Google Scholar
  11. Kopatz I, Remy JS, Behr JP (2004) A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin. J Gene Med 6:769–776CrossRefGoogle Scholar
  12. Labat-Moleur F, Steffan AM, Brisson C, Perron H, Feugeas O, Furstenberger P, Oberling F, Brambilla E, Behr JP (1996) An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther 3:1010–1017Google Scholar
  13. Leshchyns’ka I, Sytnyk V, Richter M, Andreyeva A, Puchkov D, Schachner M (2006) The adhesion molecule CHL1 regulates uncoating of clathrin-coated synaptic vesicles. Neuron 52:1011–1025CrossRefGoogle Scholar
  14. Mislick KA, Baldeschwieler JD (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci U S A 93:12349–12354CrossRefGoogle Scholar
  15. Morris MC, Depollier J, Mery J, Heitz F, Divita G (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat Biotechnol 19:1173–1176CrossRefGoogle Scholar
  16. Prochiantz A (2000) Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 12:400–406CrossRefGoogle Scholar
  17. Raptis LH, Liu SK, Firth KL, Stiles CD, Alberta JA (1995) Electroporation of peptides into adherent cells in situ. Biotechniques 18:104, 106, 108, 110 passimGoogle Scholar
  18. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 278:585–590CrossRefGoogle Scholar
  19. Saalik P, Elmquist A, Hansen M, Padari K, Saar K, Viht K, Langel U, Pooga M (2004) Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjug Chem 15:1246–1253CrossRefGoogle Scholar
  20. Schwarze SR, Dowdy SF (2000) In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci 21:45–48CrossRefGoogle Scholar
  21. Tinsley JH, Hawker J, Yuan Y (1998) Efficient protein transfection of cultured coronary venular endothelial cells. Am J Physiol 275:H1873–H1878Google Scholar
  22. Ying M, Chen B, Tian Y, Hou Y, Li Q, Shang X, Sun J, Cheng H, Zhou R (2007) Nuclear import of human sexual regulator DMRT1 is mediated by importin-beta. Biochim Biophys Acta 1773:804–813CrossRefGoogle Scholar
  23. Zelphati O, Wang Y, Kitada S, Reed JC, Felgner PL, Corbeil J (2001) Intracellular delivery of proteins with a new lipid-mediated delivery system. J Biol Chem 276:35103–35110CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Claire O. Weill
    • 1
  • Stéphanie Biri
    • 1
  • Abdennaji Adib
    • 1
  • Patrick Erbacher
    • 1
  1. 1.Polyplus-transfection, R&D DepartmentIllkirch CedexFrance

Personalised recommendations