Abstract
The central hallmark of prion diseases is the misfolding of cellular prion protein (PrPC) into a disease-associated aggregated isoform known as scrapie prion protein (PrPSc). NMR spectroscopy has made many essential contributions to the characterization of recombinant PrP in its folded, unfolded and aggregated states. Recent studies reporting on de novo generation of prions from recombinant PrP and infection of animals using prion aerosols suggest that adjustment of current biosafety measures may be necessary, particularly given the relatively high protein concentrations required for NMR applications that favor aggregation. We here present a protocol for the production of recombinant PrP under biosafety level 2 conditions that avoids entirely exposure of the experimenter to aerosols that might contain harmful PrP aggregates. In addition, we introduce an NMR sample tube setup that allows for safe handling of PrP samples at the spectrometer that usually is not part of a dedicated biosafety level 2 laboratory.
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References
Abskharon RN et al (2012) A novel expression system for production of soluble prion proteins in E. coli. Microb Cell Fact 11:6
Brown P et al (2000) Iatrogenic Creutzfeldt-Jakob disease at the millennium. Neurology 55:1075–1081
Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS (1991) Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry 30:7672–7680
Cobb NJ, Apetri AC, Surewicz WK (2008) Prion protein amyloid formation under native-like conditions involves refolding of the C-terminal alpha-helical domain. J Biol Chem 283:34704–34711
De Marco A (1977) pH dependence of internal references. J Magn Reson 26:527–528
Denkers ND, Seelig DM, Telling GC, Hoover EA (2010) Aerosol and nasal transmission of chronic wasting disease in cervidized mice. J Gen Virol 91:1651–1658
Denkers ND et al (2013) Aerosol transmission of chronic wasting disease in white-tailed deer. J Virol 87:1890–1892
Fryer HR, McLean AR (2011) There is no safe dose of prions. PLoS ONE 6:e23664
Gerum C, Silvers R, Wirmer-Bartoschek J, Schwalbe H (2009) Unfolded-state structure and dynamics influence the fibril formation of human prion protein. Angewandte Chemie (International ed) 48:9452–9456
Gerum C, Schlepckow K, Schwalbe H (2010) The unfolded state of the murine prion protein and properties of single-point mutants related to human prion diseases. J Mol Biol 401:7–12
Haybaeck J et al (2011) Aerosols transmit prions to immunocompetent and immunodeficient mice. PLoS Pathog 7:e1001257
Hornemann S, Glockshuber R (1996) Autonomous and reversible folding of a soluble amino-terminally truncated segment of the mouse prion protein. J Mol Biol 261:614–619
Kumar J et al (2010) Prion protein amyloid formation involves structural rearrangements in the C-terminal domain. ChemBioChem 11:1208–1213
Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB (2004) Synthetic mammalian prions. Science 305:673–676
Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, Mackenzie J, Will RG (2004) Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion. Lancet 363:417–421
Lysek DA et al (2005) Prion protein NMR structures of cats, dogs, pigs, and sheep. Proc Natl Acad Sci USA 102:640–645
Makarava N, Baskakov IV (2008) Expression and purification of full-length recombinant PrP of high purity. Methods Mol Biol 459:131–143
Makarava N et al (2010) Recombinant prion protein induces a new transmissible prion disease in wild-type animals. Acta Neuropathol 119:177–187
Mehlhorn I et al (1996) High-level expression and characterization of a purified 142-residue polypeptide of the prion protein. Biochemistry 35:5528–5537
Mori S, Abeygunawardana C, O’Neil Johnson M, Van Zijl PCM (1995) Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. J Magn Reson B 108:94–98
Pan KM et al (1993) Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 90:10962–10966
Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW (2004) Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 364:527–529
Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95:13363–13383
Rezaei H et al (2000) High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility. Eur J Biochem/FEBS 267:2833–2839
Riek R, Hornemann S, Wider G, Billeter M, Glockshuber R, Wuthrich K (1996) NMR structure of the mouse prion protein domain PrP(121-231). Nature 382:180–182
Schlepckow K, Schwalbe H (2013) Molecular mechanism of prion protein oligomerization at atomic resolution. Angewandte Chemie (International ed) 52:10002–10005
Schutzmaßnahmen bei Tätigkeiten mit Transmissibler Spongiformer Enzephalopathie (TSE) assoziierter Agenzien in TSE-Laboratorien. Beschluss 603 des Ausschusses für Biologische Arbeitsstoffe (2011)
Silveira JR, Raymond GJ, Hughson AG, Race RE, Sim VL, Hayes SF, Caughey B (2005) The most infectious prion protein particles. Nature 437:257–261
Tycko R, Savtchenko R, Ostapchenko VG, Makarava N, Baskakov IV (2010) The α-helical C-terminal domain of full-length recombinant PrP converts to an in-register parallel β-sheet structure in PrP fibrils: evidence from solid state nuclear magnetic resonance. Biochemistry 49:9488–9497
Walsh P, Simonetti K, Sharpe S (2009) Core structure of amyloid fibrils formed by residues 106–126 of the human prion protein. Structure 17:417–426
Wang F, Wang X, Yuan CG, Ma J (2010) Generating a prion with bacterially expressed recombinant prion protein. Science 327:1132–1135
Ward HJ et al (2006) Risk factors for variant Creutzfeldt-Jakob disease: a case-control study. Ann Neurol 59:111–120
Will RG et al (1996) A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347:921–925
World Health Organization. (1999) WHO infection control guidelines for transmissible spongiform encephalopathies. WHO, Geneva, Switzerland
Zahn R, von Schroetter C, Wuthrich K (1997) Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding. FEBS Lett 417:400–404
Zahn R et al (2000) NMR solution structure of the human prion protein. Proc Natl Acad Sci USA 97:145–150
Acknowledgments
We gratefully acknowledge advice and support from Dr. Edgar Holznagel (Paul-Ehrlich-Institut, Langen, Germany) and Dr. Monika Schneider and Dr. Heike Körber (both Biological Safety, Goethe University Frankfurt) in devising and establishing the herein described procedures. Financial support of this work through the German Research Foundation (DFG: Schw701/9-1, 9-2) and through the state of Hesse (BMRZ) is gratefully acknowledged. H.S. is a member of the DFG-funded Cluster of Excellence: Macromolecular Complexes.
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The authors declare that they have no conflict of interest.
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Peter Rehbein and Kai Schlepckow have contributed equally to this work.
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Rehbein, P., Saxena, K., Schlepckow, K. et al. Protocol for aerosol-free recombinant production and NMR analysis of prion proteins. J Biomol NMR 59, 111–117 (2014). https://doi.org/10.1007/s10858-014-9831-5
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DOI: https://doi.org/10.1007/s10858-014-9831-5