Amino Acids

, Volume 46, Issue 10, pp 2415–2426 | Cite as

A routine method for cloning, expressing and purifying Aβ(1–42) for structural NMR studies

  • Daniel K. Weber
  • Marc-Antoine Sani
  • John D. GehmanEmail author
Original Article


Nuclear magnetic resonance (NMR) is a key technology in the biophysicist’s toolbox for gaining atomic-level insight into structure and dynamics of biomolecules. Investigation of the amyloid-β peptide (Aβ) of Alzheimer’s disease is one area where NMR has proven useful, and holds even more potential. A barrier to realizing this potential, however, is the expense of the isotopically enriched peptide required for most NMR work. Whereas most biomolecular NMR studies employ biosynthetic methods as a very cost-effective means to obtain isotopically enriched biomolecules, this approach has proven less than straightforward for Aβ. Furthermore, the notorious propensity of Aβ to aggregate during purification and handling reduces yields and increases the already relatively high costs of solid phase synthesis methods. Here we report our biosynthetic and purification developments that yield pure, uniformly enriched 15N and 13C15N Aβ(1–42), in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods, which we characterize by solution-state NMR, thioflavin T assays, circular dichroism, electrospray mass spectrometry, and dynamic light scattering. These developments should facilitate further investigations into Alzheimer’s disease, and perhaps misfolding diseases in general.


Recombinant peptide Uniform labeling Amyloid beta peptide Alzheimer’s disease Protein NMR SUMO Thioflavin T assay Circular dichroism 


Amyloid beta peptide


Amyloid precursor protein


Circular dichroism


Column volume


Dynamic light scattering


Electrospray ionization mass spectrometry


Guanidine hydrochloride


Green fluorescent protein


Heteronuclear single quantum coherence


Isopropyl β-d-1-thiogalactopyranoside


Luria Broth


Liquid chromatography-mass spectrometry


Methionine aminopeptidase


Nuclear magnetic resonance


Nickel-nitrilotriacetic acid


Post-translational modification


Reactive oxygen species


Reverse-phase high-performance liquid chromatography


Room temperature


Solid phase peptide synthesis


Small ubiquitin-like modifier


Terrific broth


Tobacco etch virus protease




Ulb-specific protease 1



The authors would like to sincerely thank Dr. Nick Williamson, Paul O’Donnell and Michael Leeming for discussions regarding ESI-MS acquisition and analysis, John Karas for advice on HPLC purification, and Professor Anthony Wedd and Dr. Zhiguang Xiao for allowing access to equipment required for cell-culture work. J. Gehman was partially funded by ARC Future Fellowship FT0991558 for this work. Circular Dichroism and Dynamic Light Scattering instruments were funded by a LIEF grant LE120100186 to G. Bryant (RMIT) and J. Gehman. D. Weber is thankful for an Australian Postgraduate Award PhD scholarship and Dowd Foundation Postgraduate Research Scholarship for Neuroscience.

Conflict of Interest

The authors declare no conflict of interest.


  1. Ball KA, Phillips AH, Wemmer DE, Head-Gordon T (2013) Differences in β-strand populations of monomeric Aβ40 and Aβ42. Biophys J 104:2714–2724PubMedCrossRefPubMedCentralGoogle Scholar
  2. Barnham KJ, Ciccotosto GD, Tickler AK, Ali FE, Smith DG, Williamson NA, Lam YH, Carrington D, Tew D, Kocak G, Volitakis I, Separovic F, Barrow CJ, Wade JD, Masters CL, Cherny RA, Curtain CC, Bush AI, Cappai R (2003) Neurotoxic, redox-competent Alzheimer’s β-amyloid is released from lipid membrane by methionine oxidation. J Biol Chem 278:42959–42965PubMedCrossRefGoogle Scholar
  3. Barnham KJ, Haeffner F, Ciccotosto GD, Curtain CC, Tew D, Mavros C, Beyreuther K, Carrington D, Masters CL, Cherny RA, Cappai R, Bush AI (2004) Tyrosine gated electron transfer is key to the toxic mechanism of Alzheimer’s disease β-amyloid. FASEB J 18:1427–1429PubMedGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Broersen K, Jonckheere W, Rozenski J, Vandersteen A, Pauwels K, Pastore A, Rousseau F, Schymkowitz J (2011) A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer’s disease. Protein Eng Des Sel 24:743–750PubMedCrossRefGoogle Scholar
  6. Butterfield DA, Lauderback CM (2002) Lipid peroxidation and protein oxidation in Alzheimer’s disease brain: potential causes and consequences involving amyloid β-peptide-associated free radical oxidative stress. Free Radic Biol Med 32:1050–1060PubMedCrossRefGoogle Scholar
  7. Coles M, Bicknell W, Watson RA, Fairlie DP, Craik DJ (1998) Solution structure of amyloid β-peptide(1–40) in a water-micelle environment. Is the membrane-spanning domain where we think it is? Biochemistry 37:11064–11077PubMedCrossRefGoogle Scholar
  8. Crescenzi O, Tomaselli S, Guerrini R, Salvadori S, D’Ursi AM, Temussi PA, Picone D (2002) Solution structure of the Alzheimer amyloid β-peptide (1–42) in an apolar microenvironment: similarity with a virus fusion domain. Eur J Biochem 269:5642–5648PubMedCrossRefGoogle Scholar
  9. Danielsson J, Andersson A, Jarvet J, Gräslund A (2006) 15 N relaxation study of the amyloid β-peptide: structural propensities and persistence length. Magn Reson Chem 44:S114–S121PubMedCrossRefGoogle Scholar
  10. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293PubMedCrossRefGoogle Scholar
  11. Finder VH, Vodopivec I, Nitsch RM, Glockshuber R (2010) The recombinant amyloid-β peptide Aβ1–42 aggregates faster and is more neurotoxic than synthetic Aβ1–42. J Mol Biol 396:9–18PubMedCrossRefGoogle Scholar
  12. Frottin F, Martinez A, Peynot P, Mitra S, Holz RC, Giglione C, Meinnel T (2006) The proteomics of N-terminal methionine cleavage. Mol Cell Proteomics 5:2336–2349PubMedCrossRefGoogle Scholar
  13. Gehman JD, O’Brien CC, Shabanpoor F, Wade JD, Separovic F (2008a) Metal effects on the membrane interactions of amyloid-β peptides. Eur Biophys J 37:333–344PubMedCrossRefGoogle Scholar
  14. Gehman JD, Cocco MJ, Grindley NDF (2008b) Chemical shift mapping of γδ resolvase dimer and activated tetramer: mechanistic implications for DNA strand exchange. Biochimica et Biophysica Acta-Proteins Proteomics 1784:2086–2092CrossRefGoogle Scholar
  15. Geoghegan KF, Dixon HBF, Rosner PJ, Hoth LR, Lanzetti AJ, Borzilleri KA, Marr ES, Pezzullo LH, Martin LB, Lemotte PK, McColl AS, Kamath AV, Stroh JG (1999) Spontaneous α-N-6-phosphogluconoylation of a ‘His tag’ in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins. Anal Biochem 267:169–184PubMedCrossRefGoogle Scholar
  16. Ghalebani L, Wahlström A, Danielsson J, Wärmländer SKTS, Gräslund A (2012) PH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide. Biochem Biophys Res Commun 421:554–560PubMedCrossRefGoogle Scholar
  17. Hortschansky P, Schroeckh V, Christopeit T, Zandomeneghi G, Fändrich M (2005) The aggregation kinetics of Alzheimer’s β-amyloid peptide is controlled by stochastic nucleation. Protein Sci 14:1753–1759PubMedCrossRefPubMedCentralGoogle Scholar
  18. Hou L, Shao H, Zhang Y, Li H, Menon NK, Neuhaus EB, Brewer JM, Byeon IJL, Ray DG, Vitek MP, Iwashita T, Makula RA, Przybyla AB, Zagorski MG (2004) Solution NMR studies of the Aβ(1–40) and Aβ(1–42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation. J Am Chem Soc 126:1992–2005PubMedCrossRefGoogle Scholar
  19. Kollipara L, Zahedi RP (2013) Protein carbamylation: in vivo modification or in vitro artefact? Proteomics 13:941–944PubMedCrossRefGoogle Scholar
  20. Kuipers BJH, Gruppen H (2007) Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J Agric Food Chem 55:5445–5451PubMedCrossRefGoogle Scholar
  21. Lau TL, Gehman JD, Wade JD, Masters CL, Barnham KJ, Separovic F (2007a) Cholesterol and clioquinol modulation of Aβ(1–42) interaction with phospholipid bilayers and metals. Biochimica et Biophysica Acta-Biomembr 1768:3135–3144CrossRefGoogle Scholar
  22. Lau TL, Gehman JD, Wade JD, Perez K, Masters CL, Barnham KJ, Separovic F (2007b) Membrane interactions and the effect of metal ions of the amyloidogenic fragment Aβ(25–35) in comparison to Aβ(1–42). Biochimica et Biophysica Acta-Biomembr 1768:2400–2408CrossRefGoogle Scholar
  23. Lee EK, Hwang JH, Shin DY, Kim DI, Yoo YJ (2005) Production of recombinant amyloid-β peptide 42 as an ubiquitin extension. Protein Expr Purif 40:183–189PubMedCrossRefGoogle Scholar
  24. Lee CD, Sun HC, Hu SM, Chiu CF, Homhuan A, Liang SM, Leng CH, Wang TF (2008) An improved SUMO fusion protein system for effective production of native proteins. Protein Sci 17:1241–1248PubMedCrossRefPubMedCentralGoogle Scholar
  25. Long F, Cho W, Ishii Y (2011) Expression and purification of 15 N- and 13C-isotope labeled 40-residue human Alzheimer’s β-amyloid peptide for NMR-based structural analysis. Protein Expr Purif 79:16–24PubMedCrossRefPubMedCentralGoogle Scholar
  26. Lu JX, Qiang W, Yau WM, Schwieters CD, Meredith SC, Tycko R (2013) Molecular structure of β-amyloid fibrils in Alzheimer’s disease brain tissue. Cell 154:1257–1268PubMedCrossRefGoogle Scholar
  27. Lührs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Döbeli H, Schubert D, Riek R (2005) 3D structure of Alzheimer’s amyloid-β(1–42) fibrils. Proc Natl Acad Sci USA 102:17342–17347PubMedCrossRefPubMedCentralGoogle Scholar
  28. Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics 5:75–86PubMedCrossRefGoogle Scholar
  29. Mehta AK, Rosen RF, Childers WS, Gehman JD, Walker LC, Lynn DG (2013) Context dependence of protein misfolding and structural strains in neurodegenerative diseases. Biopolymers 100:722–730PubMedCrossRefGoogle Scholar
  30. Nagata-Uchiyama M, Yaguchi M, Hirano Y, Ueda T (2007) Expression and purification of uniformly 15 N-labeled amyloid β peptide 1–40 in Escherichia coli. Protein Pept Lett 14:788–792PubMedCrossRefGoogle Scholar
  31. Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747PubMedPubMedCentralGoogle Scholar
  32. Paravastu AK, Leapman RD, Yau WM, Tycko R (2008) Molecular structural basis for polymorphism in Alzheimer’s β-amyloid fibrils. Proc Natl Acad Sci USA 105:18349–18354PubMedCrossRefPubMedCentralGoogle Scholar
  33. Petkova AT, Ishii Y, Balbach JJ, Antzutkin ON, Leapman RD, Delaglio F, Tycko R (2002) A structural model for Alzheimer’s β-amyloid fibrils based on experimental constraints from solid state NMR. Proc Natl Acad Sci USA 99:16742–16747PubMedCrossRefPubMedCentralGoogle Scholar
  34. Petkova AT, Yau WM, Tycko R (2006) Experimental constraints on quaternary structure in Alzheimer’s β-amyloid fibrils. Biochemistry 45:498–512PubMedCrossRefPubMedCentralGoogle Scholar
  35. Rezaei-Ghaleh N, Andreetto E, Yan LM, Kapurniotu A, Zweckstetter M (2011) Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide. PLoS One 6:e20289Google Scholar
  36. Rosenman DJ, Connors CR, Chen W, Wang C, García AE (2013) Aβ monomers transiently sample oligomer and fibril-like configurations: ensemble characterization using a combined MD/NMR approach. J Mol Biol 425:3338–3359PubMedCrossRefGoogle Scholar
  37. Ryan TM, Caine J, Mertens HDT, Kirby N, Nigro J, Breheney K, Waddington LJ, Streltsov VA, Curtain C, Masters CL, Roberts BR (2013) Ammonium hydroxide treatment of Aβ produces an aggregate free solution suitable for biophysical and cell culture characterization. Peer J 1:e73PubMedCrossRefPubMedCentralGoogle Scholar
  38. Sani MA, Gehman JD, Separovic F (2011) Lipid matrix plays a role in Abeta fibril kinetics and morphology. FEBS Lett 585:749–754PubMedCrossRefGoogle Scholar
  39. Satakarni M, Curtis R (2011) Production of recombinant peptides as fusions with SUMO. Protein Expr Purif 78:113–119PubMedCrossRefGoogle Scholar
  40. Sciacca MFM, Kotler SA, Brender JR, Chen J, Lee DK, Ramamoorthy A (2012) Two-step mechanism of membrane disruption by Aβ through membrane fragmentation and pore formation. Biophys J 103:702–710PubMedCrossRefPubMedCentralGoogle Scholar
  41. Selkoe DJ (2012) Preventing Alzheimer’s disease. Science 337:1488–1492PubMedCrossRefGoogle Scholar
  42. Sgourakis NG, Merced-Serrano M, Boutsidis C, Drineas P, Du Z, Wang C, Garcia AE (2011) Atomic-level characterization of the ensemble of the Aβ(1–42) monomer in water using unbiased molecular dynamics simulations and spectral algorithms. J Mol Biol 405:570–583PubMedCrossRefPubMedCentralGoogle Scholar
  43. Shahnawaz M, Thapa A, Park IS (2007) Stable activity of a deubiquitylating enzyme (Usp2-cc) in the presence of high concentrations of urea and its application to purify aggregation-prone peptides. Biochem Biophys Res Commun 359:801–805PubMedCrossRefGoogle Scholar
  44. Sisodia SS (1992) β-Amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci USA 89:6075–6079PubMedCrossRefPubMedCentralGoogle Scholar
  45. Sticht H, Bayer P, Willbold D, Dames S, Hilbich C, Beyreuther K, Frank RW, Rosch P (1995) Structure of amyloid A4-(1–40)-peptide of Alzheimer’s disease. Eur J Biochem 233:293–298PubMedCrossRefGoogle Scholar
  46. Thapa A, Shahnawaz M, Karki P, Dahal GR, Sharoar MG, Shin SY, Lee JS, Cho B, Park IS (2008) Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins. Biotechniques 44:787–796PubMedCrossRefGoogle Scholar
  47. Tomaselli S, Esposito V, Vangone P, Van Nuland NAJ, Bonvin AMJJ, Guerrini R, Tancredi T, Temussi PA, Picone D (2006) The α-to-β conformational transition of Alzheimer’s Aβ-(1–42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of β conformation seeding. Chem Bio Chem 7:257–267PubMedCrossRefGoogle Scholar
  48. Vivekanandan S, Brender JR, Lee SY, Ramamoorthy A (2011) A partially folded structure of amyloid-beta(1–40) in an aqueous environment. Biochem Biophys Res Commun 411:312–316PubMedCrossRefPubMedCentralGoogle Scholar
  49. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins: Structure. Funct Genet 59:687–696CrossRefGoogle Scholar
  50. Walsh DM, Thulin E, Minogue AM, Gustavsson N, Pang E, Teplow DB, Linse S (2009) A facile method for expression and purification of the Alzheimer’s disease-associated amyloid β-peptide. FEBS J 276:1266–1281PubMedCrossRefPubMedCentralGoogle Scholar
  51. Watson AA, Fairlie DP, Craik DJ (1998) Solution structure of methionine-oxidized amyloid β-peptide (1–40). Does oxidation affect conformational switching? Biochemistry 37:12700–12706PubMedCrossRefGoogle Scholar
  52. Watt AD, Villemagne VL, Barnham KJ (2012) Metals, membranes, and amyloid-β oligomers: key pieces in the Alzheimer’s disease puzzle? In: Perry G, Zhu X, Smith MA, Sorensen A, Avila J (eds), 3rd edn. 33:S283–S293Google Scholar
  53. Weber DK, Gehman JD, Separovic F, Sani MA (2012) Copper modulation of amyloid beta 42 interactions with model membranes. Aust J Chem 65:472–479CrossRefGoogle Scholar
  54. Williamson MP, Suzuki Y, Bourne NT, Asakura T (2006) Binding of amyloid β-peptide to ganglioside micelles is dependent on histidine-13. Biochem J 397:483–490PubMedCrossRefPubMedCentralGoogle Scholar
  55. Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson HJ, Oldfield E, Markley JL, Sykes BD (1995) 1H, 13C and 15 N chemical shift referencing in biomolecular NMR. J Biomol NMR 6:135–140PubMedCrossRefGoogle Scholar
  56. Yan Y, McCallum SA, Wang C (2008) M35 oxidation induces Aβ40-like structural and dynamical changes in Aβ42. J Am Chem Soc 130:5394–5395PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Daniel K. Weber
    • 1
  • Marc-Antoine Sani
    • 1
  • John D. Gehman
    • 1
    • 2
    Email author
  1. 1.School of Chemistry, Bio21 InstituteUniversity of MelbourneMelbourneAustralia
  2. 2.GehmanLabWoodendAustralia

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