Synthesis of Cell-Penetrating Peptides and Their Application in Neurobiology

  • Gunnar P. H. Dietz
  • Mathias Bähr
Part of the Methods in Molecular Biology book series (MIMB, volume 399)


Short basic amino acid sequences, often called cell-penetrating peptides (CPPs), allow the delivery of proteins and other molecules into cells and across the blood-brain barrier (BBB). Although the ability of basic proteins to facilitate such trafficking is known for a long time, only the application of genetic methods and overexpression of fusion proteins in Escherichia coli has lead to a wide application of CPP in many research areas, including signal transduction, cancer, angiogenesis, apoptosis, bone development, cardioprotection, cell cycle, neurobiology, and many others. For the neuroscientist, CPPs are particularly attractive, as a number of articles in the last 5 years have reported their use for neuronal rescue in a number of models for neurodegenerative diseases in vitro and in vivo in rats, mice, or gerbils. Here, we give a detailed description of the protein purification methodology and applications in neuroscience.

Key Words

Protein delivery Trojan horse peptide arginine-rich peptide ischemia trauma apoptosis excitotoxicity cell-penetrating peptides (CPP) protein transduction domain (PTD) transactivator of transcription (Tat) 


  1. 1.
    Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L.L., Pepinsky, B., Barsoum, J. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci USA 91, 664–8.CrossRefPubMedGoogle Scholar
  2. 2.
    Nagahara, H., Vocero-Akbani, A.M., Snyder, E.L., Ho, A., Latham, D.G., Lissy, N.A., Becker-Hapak, M., Ezhevsky, S.A., Dowdy, S.F. (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med 4, 1449–52.CrossRefPubMedGoogle Scholar
  3. 3.
    Schwarze, S.R., Ho, A., Vocero-Akbani, A., Dowdy, S.F. (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285, 1569–72.CrossRefPubMedGoogle Scholar
  4. 4.
    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, 1246–53.CrossRefPubMedGoogle Scholar
  5. 5.
    Lin, Q., Jo, D., Grebre-Amlak, K.D., Ruley, H.E. (2004) Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells. BMC Biotechnol 4, 25.CrossRefPubMedGoogle Scholar
  6. 6.
    Dietz, G.P.H., Kilic, E., Bähr, M. (2004) Delivery of bioactive molecules into the cell: the Trojan Horse approach. Mol Cell Neurosci 27, 85–131.CrossRefPubMedGoogle Scholar
  7. 7.
    Dietz, G.P.H., Bähr, M. ((2005) PPeptide-enhanced cellular internalization of proteins in neuroscience. Brain Res Bull 68(1–2), 103–14.CrossRefPubMedGoogle Scholar
  8. 8.
    Allinquant, B., Hantraye, P., Mailleux, P., Moya, K., Bouillot, C., Prochiantz, A. (1995) Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro. J Cell Biol 128, 919–27.CrossRefPubMedGoogle Scholar
  9. 9.
    Pizzi, M., Sarnico, I., Boroni, F., Benarese, M., Steimberg, N., Mazzoleni, G., Dietz, G.P.H., Bähr, M., Liou, H.C., Spano, P.F. (2005) NF-kappaB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity. Cell Death Differ 12, 761–72.CrossRefPubMedGoogle Scholar
  10. 10.
    Troy, C.M., Stefanis, L., Prochiantz, A., Greene, L.A., Shelanski, M.L. (1996) The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation. Proc Natl Acad Sci USA 93, 5635–40.CrossRefPubMedGoogle Scholar
  11. 11.
    Liu, X.H., Castelli, J.C., Youle, R.J. (1999) Receptor-mediated uptake of an extracellular Bcl-x(L) fusion protein inhibits apoptosis. Proc Natl Acad Sci USA 96, 9563–7.CrossRefPubMedGoogle Scholar
  12. 12.
    Wu, H.Y., Tomizawa, K., Matsushita, M., Lu, Y.F., Li, S.T., Matsui, H. (2003) Poly-arginine-fused calpastatin peptide, a living cell membrane-permeable and specific inhibitor for calpain. Neurosci Res 47, 131–5.CrossRefPubMedGoogle Scholar
  13. 13.
    Kilic, E., Dietz, G.P.H., Hermann, D.M., Bähr, M. ((2002) Intravenous TAT-Bcl-XL is protective after middle cerebral artery occlusion in mice. Ann Neurol 52, 617–22.CrossRefPubMedGoogle Scholar
  14. 14.
    Cao, G., Pei, W., Ge, H., Liang, Q., Luo, Y., Sharp, F.R., Lu, A., Ran, R., Graham, S.H., Chen, J. (2002) In vivo delivery of a Bcl-xL fusion protein containing the Tat protein transduction domain protects against ischemic brain injury and neuronal apoptosis. J Neurosci 22, 5423–31.PubMedGoogle Scholar
  15. 15.
    Dietz, G.P.H., Kilic, E., Bähr, M. (2002) Inhibition of neuronal apoptosis in vitro and in vivo using TAT-mediated protein transduction. Mol Cell Neurosci 21, 29–37.CrossRefPubMedGoogle Scholar
  16. 16.
    Asoh, S., Ohsawa, I., Mori, T., Katsura, K., Hiraide, T., Katayama, Y., Kimura, M., Ozaki, D., Yamagata, K., Ohta, S. (2002) Protection against ischemic brain injury by protein therapeutics. Proc Natl Acad Sci USA 99, 17107–12.CrossRefPubMedGoogle Scholar
  17. 17.
    Theodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P., Prochiantz, A. (1995) Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. J Neurosci 15, 7158–67.PubMedGoogle Scholar
  18. 18.
    Bertrand, J., McKerracher, L.J. (2003) Intravitreal injection of C3–05 promotes regeneration after intraorbital microlesion of the optic nerve in adult rat. Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Program No. 678.11.Google Scholar
  19. 19.
    Winton, M.J., Dubreuil, C.I., Campenot, R.B., McKerracher, L. (2003) The effects of C3–05 treatment on sympathetic neurons plated in Campenot chambers. Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Program No. 678.7.Google Scholar
  20. 20.
    Bertrand, J., Winton, M.J., Rodriguez-Hernandez, N., Campenot, R.B., McKerracher, L. (2005) Application of Rho antagonist to neuronal cell bodies promotes neurite growth in compartmented cultures and regeneration of retinal ganglion cell axons in the optic nerve of adult rats. J. Neurosci. 25, 1113–21.CrossRefPubMedGoogle Scholar
  21. 21.
    Asanuma, T., Inanami, O., Tabu, K., Waki, K., Kon, Y., Kuwabara, M. (2004) Protection against malonate-induced ischemic brain injury in rat by a cell-permeable peptidic c-Jun N-terminal kinase inhibitor, (L)-HIV-TAT48-57-PP-JBD20, observed by the apparent diffusion coefficient mapping magnetic resonance imaging method. Neurosci Lett 359, 57–60.CrossRefPubMedGoogle Scholar
  22. 22.
    Borsello, T., Clarke, P.G., Hirt, L., Vercelli, A., Repici, M., Schorderet, D.F., Bogousslavsky, J., Bonny, C. (2003) A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat Med 9, 1180–6.CrossRefPubMedGoogle Scholar
  23. 23.
    Kilic, ü., Kilic, E., Dietz, G.P.H., Bähr, M. ((2003) Intravenous TAT-GDNF is protective after focal cerebral ischemia in mice. Stroke 34, 1304–10.CrossRefPubMedGoogle Scholar
  24. 24.
    Aarts, M., Liu, Y., Liu, L., Besshoh, S., Arundine, M., Gurd, J.W., Wang, Y.T., Salter, M.W., Tymianski, M. (2002) Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science 298, 846–50.CrossRefPubMedGoogle Scholar
  25. 25.
    Pardridge, W.M. (2002) Blood-brain barrier drug targeting enables neuroprotection in brain ischemia following delayed intravenous administration of neurotrophins. Adv Exp Med Biol 513, 397–430.PubMedGoogle Scholar
  26. 26.
    Song, B.W., Vinters, H.V., Wu, D., Pardridge, W.M. (2002) Enhanced neuroprotective effects of basic fibroblast growth factor in regional brain ischemia after conjugation to a blood-brain barrier delivery vector. J Pharmacol Exp Ther 301, 605–10.CrossRefPubMedGoogle Scholar
  27. 27.
    Eum, W.S., Won Kim, D., Koo Hwang, I., Yoo, K.Y., Kang, T.C., Ho Jang, S., Soon Choi, H., Hyun Choi, S., Hoon Kim, Y., Young Kim, S., Yil Kwon, H., Hoon Kang, J., Kwon, O.S., Cho, S.W., Soo Lee, K., Park, J., Ho Won, M., Young Choi, S. (2004) In vivo protein transduction: biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult. Free Radic Biol Med 37, 1656–69.CrossRefPubMedGoogle Scholar
  28. 28.
    Diem, R., Taheri, N., Dietz, G.P.H., Kuhnert, A., Maier, K., Sättler, M.B., Gadjanski, I., Merkler, D., Bähr, M. (2(2005) HIHIV-Tat-mediated Bcl-xL delivery protects retinal ganglion cells during experimental autoimmune optic neuritis. Neurobiol Dis 20, 218–26.CrossRefPubMedGoogle Scholar
  29. 29.
    Blum, S., Dash, P.K. (2004) A cell-permeable phospholipase Cgamma1-binding peptide transduces neurons and impairs long-term spatial memory. Learn Mem 11, 239–43.CrossRefPubMedGoogle Scholar
  30. 30.
    Matsushita, M., Tomizawa, K., Moriwaki, A., Li, S.T., Terada, H., Matsui, H. (2001) A high-efficiency protein transduction system demonstrating the role of PKA in long-lasting long-term potentiation. J Neurosci 21, 6000–7.PubMedGoogle Scholar
  31. 31.
    Elliger, S.S., Elliger, C.A., Lang, C., Watson, G.L. (2002) Enhanced secretion and uptake of beta-glucuronidase improves adeno-associated viral-mediated gene therapy of mucopolysaccharidosis type VII mice. Mol Ther 5, 617–26.CrossRefPubMedGoogle Scholar
  32. 32.
    Xia, H., Mao, Q., Davidson, B.L. (2001) The HIV Tat protein transduction domain improves the biodistribution of beta-glucuronidase expressed from recombinant viral vectors. Nat Biotechnol 19, 640–4.CrossRefPubMedGoogle Scholar
  33. 33.
    Kelemen, B.R., Hsiao, K., Goueli, S.A. (2002) Selective in vivo inhibition of mitogen-activated protein kinase activation using cell-permeable peptides. J Biol Chem 277, 8741–8.CrossRefPubMedGoogle Scholar
  34. 34.
    Williams, E.J., Doherty, P. (1999) Evidence for and against a pivotal role of PI 3-kinase in a neuronal cell survival pathway. Mol Cell Neurosci 13, 272–80.CrossRefPubMedGoogle Scholar
  35. 35.
    Zezula, J., Casaccia-Bonnefil, P., Ezhevsky, S.A., Osterhout, D.J., Levine, J.M., Dowdy, S.F., Chao, M.V., Koff, A. (2001) p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal. EMBO Rep 2, 27–34.CrossRefPubMedGoogle Scholar
  36. 36.
    Zhang, Y., Calon, F., Zhu, C., Boado, R.J., Pardridge, W.M. (2003) Intravenous nonviral gene therapy causes normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism. Hum Gene Ther 14, 1–12.CrossRefPubMedGoogle Scholar
  37. 37.
    Albani, D., Peverelli, E., Rametta, R., Batelli, S., Veschini, L., Negro, A., Forloni, G. (2004) Protective effect of TAT-delivered alpha-synuclein: relevance of the C-terminal domain and involvement of HSP70. FASEB J 18, 1713–5.PubMedGoogle Scholar
  38. 38.
    Ebert, S., Dietz, G.P.H., Mitchell, T.J., Michel, U., Bähr, M., Nau, R. ((2005) LLimited protection of TAT-Bcl-X(L) against pneumolysin-induced neuronal cell death. Neurosci Lett 384, 349–53.CrossRefPubMedGoogle Scholar
  39. 39.
    Kilic, ü., Kilic, E., Dietz, G.P.H., Bähr, M. (2004) The TAT protein transduction domain enhances the neuroprotective effect of GDNF after optic nerve transection. Neurodegen Dis 1, 44–49.CrossRefGoogle Scholar
  40. 40.
    Chiu, Y.L., Ali, A., Chu, C.Y., Cao, H., Rana, T.M. (2004) Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells. Chem Biol 11, 1165–75.CrossRefPubMedGoogle Scholar
  41. 41.
    Li, Y., Rosal, R.V., Brandt-Rauf, P.W., Fine, R.L. (2002) Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide. Biochem Biophys Res Commun 298, 439–49.CrossRefPubMedGoogle Scholar
  42. 42.
    Aksenov, M.Y., Hasselrot, U., Wu, G., Nath, A., Anderson, C., Mactutus, C.F., Booze, R.M. (2003) Temporal relationships between HIV-1 Tat-induced neuronal degeneration, OX-42 immunoreactivity, reactive astrocytosis, and protein oxidation in the rat striatum. Brain Res 987, 1–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Pugliese, A., Vidotto, V., Beltramo, T., Petrini, S., Torre, D. (2004) A review of HIV-1 Tat protein biological effects. Cell Biochem Funct 7, 7.Google Scholar
  44. 44.
    Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (1997) Current Protocols in Molecular Biology. John Wiley, New York.Google Scholar
  45. 45.
    Sambrook, J., Russell, D.W. (2001) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  46. 46.
    Becker-Hapak, M., McAllister, S.S., Dowdy, S.F. (2001) TAT-mediated protein transduction into mammalian cells. Methods 24, 247–56.CrossRefPubMedGoogle Scholar
  47. 47.
    Vocero-Akbani, A., Chellaiah, M.A., Hruska, K.A., Dowdy, S.F. (2001) Protein transduction: delivery of Tat-GTPase fusion proteins into mammalian cells. Methods Enzymol 332, 36–49.CrossRefPubMedGoogle Scholar
  48. 48.
    Derossi, D., Chassaing, G., Prochiantz, A. (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol 8, 84–7.CrossRefPubMedGoogle Scholar
  49. 49.
    Melan, M.A., Sluder, G. (1992) Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy. J Cell Sci 101, 731–43.PubMedGoogle Scholar
  50. 50.
    Richard, J.P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M.J., Chernomordik, L.V., Lebleu, B. (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 278, 585–90.CrossRefPubMedGoogle Scholar
  51. 51.
    Moulton, H.M., Hase, M.C., Smith, K.M., Iversen, P.L. (2003) HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev 13, 31–43.CrossRefPubMedGoogle Scholar
  52. 52.
    Peitz, M., Pfannkuche, K., Rajewsky, K., Edenhofer, F. (2002) Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc Natl Acad Sci USA 99, 4489–94.CrossRefPubMedGoogle Scholar
  53. 53.
    Hällbrink, M., Oehlke, J., Papsdorf, G., Bienert, M. (2004) Uptake of cellpenetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration. Biochim Biophys Acta 1667, 222–8.CrossRefPubMedGoogle Scholar
  54. 54.
    Schutze-Redelmeier, M.P., Gournier, H., Garcia-Pons, F., Moussa, M., Joliot, A.H., Volovitch, M., Prochiantz, A., Lemonnier, F.A. (1996) Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo. J Immunol 157, 650–5.PubMedGoogle Scholar
  55. 55.
    Snyder, E.L., Meade, B.R., Saenz, C.C., Dowdy, S.F. (2004) Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide. PLoS Biol 2, E36.CrossRefPubMedGoogle Scholar
  56. 56.
    Barka, T., Gresik, E.S., Henderson, S.C. (2004) Production of cell lines secreting TAT fusion proteins. J Histochem Cytochem 52, 469–77.PubMedGoogle Scholar
  57. 57.
    Chico, D.E., Given, R.L., Miller, B.T. (2003) Binding of cationic cell-permeable peptides to plastic and glass. Peptides 24, 3–9.CrossRefPubMedGoogle Scholar
  58. 58.
    Brink, C., Österberg, E., Holmberg, K., Tiberg, F. (1992) Using poly(ethylene imine) to graft poly(ethylene glycol) or polysaccharide to polystyrene. Colloids Surf B Biointerfaces 66, 149–156.Google Scholar
  59. 59.
    Persson, D., Thoren, P.E., Herner, M., Lincoln, P., Nordén, B. (2003) Application of a novel analysis to measure the binding of the membrane-translocating Peptide penetratin to negatively charged liposomes. Biochemistry 42, 421–9.CrossRefPubMedGoogle Scholar
  60. 60.
    Dietz, G. P. H, Valbuens, P. C., Dietz, B., Meuer, K., Müller, P., Weishanpt, J. H., Bähr, M. (2006) Application of a blood-brain bamir-penetrating from of SDNF in a more model for Parkinson’s Disease. Brain Res 11082, 61–66.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Gunnar P. H. Dietz
    • 1
  • Mathias Bähr
    • 1
  1. 1.Neurologische UniversitätsklinikGöttingenGermany

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