Skip to main content
SpringerLink
Log in

Purification of Recombinant Tau Protein and Preparation of Alzheimer-Paired Helical Filaments In Vitro

  • Protocol
Amyloid Proteins

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 299))

Abstract

The tau protein is a neuronal microtubule-associated protein. Apart of its physiological function/3-the binding to and stabilization of microtubules/3-tau is found in Alzheimer's disease brain as insoluble fibers, the so-called “paired helical filaments” (PHFs). Investigating the fundamentals of tau polymerization is indispensable for identifying inhibitory conditions or compounds preventing PHF formation, which may slow down or even stop the degeneration of neurons in Alzheimer's disease. In this chapter, we describe the methods necessary for studying the characteristics of tau polymerization to PHFs. These include: a purification protocol for recombinantly expressed tau; a general method for the polyanion induced polymerization of tau to PHFs; the quantitation of PHFs by a fluorescence-based assay; the imaging and verification of PHFs by negative stain transmission electron microscopy.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 134.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Bond, J. P., Deverin, S. P., Inouye, H., El-Agnaf, O. M., Teeter, M. M., and Kirschner, D. A. (2003) Assemblies of Alzheimer's peptides Abeta25-35 and Abeta 31-35: reverse-turn conformation and side-chain interactions revealed by X-ray diffraction. J. Struct. Biol. 141(2), 156–170.

    Article  PubMed  CAS  Google Scholar 

  2. Petkova, A. T., Ishii, Y., Balbach, J. J., et al. (2002) A structural model for Alzheimer's beta-amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl. Acad. Sci. USA 99(26), 16742–16747.

    Article  PubMed  CAS  Google Scholar 

  3. Torok, M., Milton, S., Kayed, R., et al. (2002) Structural and dynamic features of Alzheimer's Abeta peptide in amyloid fibrils studied by site-directed spin labeling. J. Biol. Chem. 277(43), 40810–40815.

    Article  PubMed  Google Scholar 

  4. Rochet, J. C. and Lansbury, P. T. Jr. (2000) Amyloid fibrillogenesis: themes and variations. Curr. Opin. Struct. Biol. 10(1), 60–68.

    Article  PubMed  CAS  Google Scholar 

  5. Garcia, M. L. and Cleveland, D. W. (2001) Going new places using an old MAP: tau, microtubules and human neurodegenerative disease. Curr. Opin. Cell Biol. 13(1), 41–48.

    Article  PubMed  CAS  Google Scholar 

  6. Goedert, M., Spillantini, M. G., and Davies, S. W. (1998) Filamentous nerve cell inclusions in neurodegenerative diseases. Curr. Opin. Neurobiol. 8(5), 619–632.

    Article  PubMed  CAS  Google Scholar 

  7. Buee, L., Bussiere, T., Buee-Scherrer, V., Delacourte, A., and Hof, P. R. (2000) Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res. Rev. 33(1), 95–130.

    Article  PubMed  CAS  Google Scholar 

  8. Reed, L. A., Wszolek, Z. K., and Hutton, M. (2001) Phenotypic correlations in FTDP-17. Neurobiol. Aging 22(1), 89–107.

    Article  PubMed  CAS  Google Scholar 

  9. Cleveland, D. W., Hwo, S. Y., and Kirschner, M. W. (1977) Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J. Mol. Biol. 116(2), 227–247.

    Article  PubMed  CAS  Google Scholar 

  10. Lee, G., Cowan, N., and Kirschner, M. (1988) The primary structure and heterogeneity of tau protein from mouse brain. Science 239, 285–288.

    Article  PubMed  CAS  Google Scholar 

  11. Schweers, O., Schonbrunn-Hanebeck, E., Marx, A., and Mandelkow, E. (1994) Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for beta-structure. J. Biol. Chem. 269(39), 24290–24297.

    PubMed  CAS  Google Scholar 

  12. Berry, R. W., Abraha, A., Lagalwar, S., et al. (2003) Inhibition of tau polymerization by its carboxy-terminal caspase cleavage fragment. Biochemistry 42(27), 8325–8331.

    Article  PubMed  CAS  Google Scholar 

  13. Minoura, K., Tomoo, K., Ishida, T., Hasegawa, H., Sasaki, M., and Taniguchi, T. (2002) Amphipathic helical behavior of the third repeat fragment in the tau microtubule-binding domain, studied by (1)H NMR spectroscopy. Biochem. Biophys. Res. Commun. 294(2), 210–214.

    Article  PubMed  CAS  Google Scholar 

  14. Esposito, G., Viglino, P., Novak, M., and Cattaneo, A. (2000) The solution structure of the C-terminal segment of tau protein. J. Pept. Sci. 6(11), 550–559.

    Article  PubMed  CAS  Google Scholar 

  15. Yanagawa, H., Chung, S. H., Ogawa, Y., et al. (1998) Protein anatomy: C-tail region of human tau protein as a crucial structural element in Alzheimer's paired helical filament formation in vitro. Biochemistry 37(7), 1979–1988.

    Article  PubMed  CAS  Google Scholar 

  16. Kirschner, D. A., Abraham, C., and Selkoe, D. J. (1986) X-ray diffraction from intraneural paired helical filaments and extraneural amyloid fibers in Alzheimer disease indicates cross-ß conformation. Proc. Natl. Acad. Sci. USA 83, 503–507.

    Article  PubMed  CAS  Google Scholar 

  17. Iqbal, K., Braak, H., Braak, E., and Grundke-Iqbal, I. (1993) Silver labeling of Alzheimer neurofibrillary changes and brain beta-amyloid. J. Histotechnology 16(N4), 335–342.

    Google Scholar 

  18. von Bergen, M., Friedhoff, P., Biernat, J., Heberle, J., Mandelkow, E. M., and Mandelkow, E. (2000) Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc. Natl. Acad. Sci. USA 97(10), 5129–5134.

    Article  Google Scholar 

  19. vonBergen, M., Barghorn, S., Li, L., et al. (2001) Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure. J. Biol. Chem. 276(51), 48165–48174.

    Google Scholar 

  20. Giannetti, A. M., Lindwall, G., Chau, M. F., Radeke, M. J., Feinstein, S. C., and Kohlstaedt, L. A. (2000) Fibers of tau fragments, but not full length tau, exhibit a cross betastructure: implications for the formation of paired helical filaments. Protein Sci. 9(12), 2427–2435.

    Article  PubMed  CAS  Google Scholar 

  21. Barghorn, S., Davies, P., and Mandelkow, E. (2004) Tau paired helical filaments from Alzheimer's disease brain and assembled in vitro are based on beta-structure in the core domain. Biochemistry 43(6), 1694–1703.

    Article  PubMed  CAS  Google Scholar 

  22. Berriman, J., Serpell, L. C., Oberg, K. A., Fink, A. L., Goedert, M., and Crowther, R. A. (2003) Tau filaments from human brain and from in vitro assembly of recombinant protein show cross-beta structure. Proc. Natl. Acad. Sci. USA 100(15), 9034–9038.

    Article  PubMed  CAS  Google Scholar 

  23. Perez, M., Valpuesta, J. M., Medina, M., Montejo de Garcini, E., and Avila, J. (1996) Polymerization of tau into filaments in the presence of heparin: the minimal sequence required for tau-tau interaction. J. Neurochem. 67(3), 1183–1190.

    Article  PubMed  CAS  Google Scholar 

  24. Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996) Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans. Nature 383(6600), 550–553.

    Article  PubMed  CAS  Google Scholar 

  25. Kampers, T., Friedhoff, P., Biernat, J., and Mandelkow, E. M. (1996) RNA stimulates aggregation of microtubule-associated protein-tau into Alzheimer-like paired helical filaments. FEBS Letters 399(3), 344–349.

    Article  PubMed  CAS  Google Scholar 

  26. Wilson, D. M. and Binder, L. I. (1997) Free fatty acids stimulate the polymerization of tau and amyloid beta peptides. In vitro evidence for a common effector of pathogenesis in Alzheimer's disease. Am. J. Pathol. 150(6), 2181–2195.

    PubMed  CAS  Google Scholar 

  27. King, M. E., Ahuja, V., Binder, L. I., and Kuret, J. (1999) Ligand-dependent tau filament formation: implications for Alzheimer's disease progression. Biochemistry 38(45), 14851–14859.

    Article  PubMed  CAS  Google Scholar 

  28. Wille, H., Drewes, G., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (1992) Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro. J. Cell Biol. 118, 573–584.

    Article  PubMed  CAS  Google Scholar 

  29. Schweers, O., Mandelkow, E. M., Biernat, J., and Mandelkow, E. (1995) Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments. Proc. Natl. Acad. Sci. USA 92(18), 8463–8467.

    Article  PubMed  CAS  Google Scholar 

  30. Friedhoff, P., Schneider, A., Mandelkow, E. M., and Mandelkow, E. (1998) Rapid assembly of Alzheimer-like paired helical filaments from microtubule-associated protein tau monitored by fluorescence in solution. Biochemistry 37(28), 10223–10230.

    Article  PubMed  CAS  Google Scholar 

  31. Foster, N. L., Wilhelmsen, K., Sima, A. A., Jones, M. Z., D'Amato, C. J., and Gilman, S. (1997) Frontotemporal dementia and parkinsonism linked to chromosome 17: a consensus conference. Ann. Neurol. 41(6), 706–715.

    Article  PubMed  CAS  Google Scholar 

  32. Hutton, M., Lendon, C. L., Rizzu, P., et al. (1998) Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393(6686), 702–705.

    Article  PubMed  CAS  Google Scholar 

  33. Clark, L. N., Poorkaj, P., Wszolek, Z., et al. (1998) Pathogenic implications of mutations in the tau gene in pallido-ponto-nigral degeneration and related neuro-degenerative disorders linked to chromosome 17. Proc. Natl. Acad. Sci. USA 95(22), 13103–13107.

    Article  PubMed  CAS  Google Scholar 

  34. Poorkaj, P., Bird, T. D., Wijsman, E., et al. (1998) Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol. 43(6), 815–825.

    Article  PubMed  CAS  Google Scholar 

  35. Goedert, M., Jakes, R., and Crowther, R. A. (1999) Effects of frontotemporal dementia FTDP-17 mutations on heparin-induced assembly of tau filaments. FEBS Lett. 450(3), 306–311.

    Article  PubMed  CAS  Google Scholar 

  36. Nacharaju, P., Lewis, J., Easson, C., et al. (1999) Accelerated filament formation from tau protein with specific FTDP-17 missense mutations. FEBS Lett. 447(2–3), 195–199.

    Article  PubMed  CAS  Google Scholar 

  37. Barghorn, S., Zheng-Fischhofer, Q., Ackmann, M., Biernat, J., von Bergen, M., and Mandelkow, E. (2000) Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias. Biochemistry 39(38), 11714–11721.

    Article  PubMed  CAS  Google Scholar 

  38. Friedhoff, P., vonBergen, M., Mandelkow, E. M., Davies, P., and Mandelkow, E. (1998) A nucleated assembly mechanism of Alzheimer paired helical filaments. Proc. Natl. Acad. Sci. USA 95(26), 15712–15717.

    Article  PubMed  CAS  Google Scholar 

  39. Wischik, C. M., Novak, M., Thogersen, H. C., et al. (1988) Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. USA 85(12), 4506–4510.

    Article  PubMed  CAS  Google Scholar 

  40. Novak, M., Kabat, J., and Wischik, C. M. (1993) Molecular characterization of the minimal protease resistant tau-unit of the Alzheimer's-disease paired helical filament. EMBO J. 12, 365–370.

    PubMed  CAS  Google Scholar 

  41. Crowther, R. A. (1991) Straight and paired helical filaments in Alzheimer disease have a common structural unit. Proc. Natl. Acad. Sci. USA 88(6), 2288–2292.

    Article  PubMed  CAS  Google Scholar 

  42. Gamblin, T. C., King, M. E., Dawson, H., et al. (2000) In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants. Biochemistry 39(20), 6136–6144.

    Article  PubMed  CAS  Google Scholar 

  43. Kidd, M. (1963) Paired helical filaments in electron microscopy of Alzheimer's disease. Nature (Lond.) 197, 192–193.

    Article  CAS  Google Scholar 

  44. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89.

    Article  PubMed  CAS  Google Scholar 

  45. Biernat, J., Mandelkow, E. M., Schröter, C., et al. (1992) The switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serine-proline motifs upstream of the microtubule binding region. EMBO J. 11, 1593–1597.

    PubMed  CAS  Google Scholar 

  46. Weingarten, M. D., Lockwood, A. H., Hwo, S. Y., and Kirschner, M. W. (1975) A protein factor essential for microtubule assembly. Proc. Natl. Acad. Sci. USA 72(5), 1858–1862.

    Article  PubMed  CAS  Google Scholar 

  47. Herzog, W. and Weber, K. (1978) Fractionation of brain microtubule-associated proteins. Isolation of two different proteins which stimulate tubulin polymerization in vitro. Eur. J. Biochem. 92(1), 1–8.

    Article  PubMed  CAS  Google Scholar 

  48. Gustke, N., Trinczek, B., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (1994) Domains of Tau protein and interactions with microtubules. Biochemistry 33, 9511–9522.

    Article  PubMed  CAS  Google Scholar 

  49. Wetlaufer, D. B. (1962) Ultraviolet spectra of proteins and amino acids. Adv. Protein Chem. 17,303–391.

    Article  CAS  Google Scholar 

  50. Saidel, L. J. and Lieberman, H. (1958) Ultraviolet absorption spectra of peptides. IV. Alanine residue. Arch. Biochem. Biophys. 76, 401–409.

    Article  PubMed  CAS  Google Scholar 

  51. Goldfarb, R. (1953) Absorption spectrum of the peptide bond. II. Influence of chain length. J. Biol. Chem. 201, 317–320.

    PubMed  CAS  Google Scholar 

  52. Barghorn, S. and Mandelkow, E. (2002) Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments. Biochemistry 41(50), 14885–14896.

    Article  PubMed  CAS  Google Scholar 

  53. Gamblin, T. C., King, M. E., Kuret, J., Berry, R. W., and Binder, L. I. (2000) Oxidative regulation of fatty acid-induced tau polymerization. Biochemistry 39(46), 14203–14210.

    Article  PubMed  CAS  Google Scholar 

  54. King, M. E., Gamblin, T. C., Kuret, J., and Binder, L. I. (2000) Differential assembly of human tau isoforms in the presence of arachidonic acid. J. Neurochem. 74(4), 1749–1757.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max-Planck-Unit for Structural Molecular Biology, Hamburg, Germany

    Stefan Barghorn, Jacek Biernat & Eckhard Mandelkow

Authors
  1. Stefan Barghorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jacek Biernat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eckhard Mandelkow
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Departments of Psychiatry and Pathology, New York University School of Medicine, New York, NY

    Einar M. Sigurdsson

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Barghorn, S., Biernat, J., Mandelkow, E. (2005). Purification of Recombinant Tau Protein and Preparation of Alzheimer-Paired Helical Filaments In Vitro. In: Sigurdsson, E.M. (eds) Amyloid Proteins. Methods in Molecular Biology™, vol 299. Humana Press. https://doi.org/10.1385/1-59259-874-9:035

Download citation

  • DOI: https://doi.org/10.1385/1-59259-874-9:035

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-337-4

  • Online ISBN: 978-1-59259-874-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation