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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
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
References
Bar-Sagi D, Feramisco JR (1985) Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell 42:841–848
Campbell PL, McCluskey J, Yeo JP, Toh BH (1995) Electroporation of antibodies into mammalian cells. Methods Mol Biol 48:83–92
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 5:2388–2397
Chuck AS, Clarke MF, Palsson BO (1996) Retroviral infection is limited by Brownian motion. Hum Gene Ther 7:1527–1534
Dalkara D, Zuber G, Behr JP (2004) Intracytoplasmic delivery of anionic proteins. Mol Ther 9:964–969
Drin G, Cottin S, Blanc E, Rees AR, Temsamani J (2003) Studies on the internalization mechanism of cationic cell-penetrating peptides. J Biol Chem 278:31192–31201
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 15:1332–1339
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 1111:239–246
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 279:12625–12635
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 61:474–477
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 6:769–776
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 3:1010–1017
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 52:1011–1025
Mislick KA, Baldeschwieler JD (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci U S A 93:12349–12354
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 19:1173–1176
Prochiantz A (2000) Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 12:400–406
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 passim
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 278:585–590
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 15:1246–1253
Schwarze SR, Dowdy SF (2000) In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci 21:45–48
Tinsley JH, Hawker J, Yuan Y (1998) Efficient protein transfection of cultured coronary venular endothelial cells. Am J Physiol 275:H1873–H1878
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 1773:804–813
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 276:35103–35110
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Weill, C.O., Biri, S., Adib, A. et al. A practical approach for intracellular protein delivery. Cytotechnology 56, 41–48 (2008). https://doi.org/10.1007/s10616-007-9102-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-007-9102-3