Abstract
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.
Similar content being viewed by others
Abbreviations
- Aβ:
-
Amyloid beta peptide
- APP:
-
Amyloid precursor protein
- CD:
-
Circular dichroism
- CV:
-
Column volume
- DLS:
-
Dynamic light scattering
- ESI-MS:
-
Electrospray ionization mass spectrometry
- Gdm.HCl:
-
Guanidine hydrochloride
- GFP:
-
Green fluorescent protein
- HSQC:
-
Heteronuclear single quantum coherence
- IPTG:
-
Isopropyl β-d-1-thiogalactopyranoside
- LB:
-
Luria Broth
- LC-MS:
-
Liquid chromatography-mass spectrometry
- MAP:
-
Methionine aminopeptidase
- NMR:
-
Nuclear magnetic resonance
- Ni–NTA:
-
Nickel-nitrilotriacetic acid
- PTM:
-
Post-translational modification
- ROS:
-
Reactive oxygen species
- RP-HPLC:
-
Reverse-phase high-performance liquid chromatography
- RT:
-
Room temperature
- SPPS:
-
Solid phase peptide synthesis
- SUMO:
-
Small ubiquitin-like modifier
- TB:
-
Terrific broth
- TEV:
-
Tobacco etch virus protease
- Ub:
-
Ubiquitin
- Ulp1:
-
Ulb-specific protease 1
References
Ball KA, Phillips AH, Wemmer DE, Head-Gordon T (2013) Differences in β-strand populations of monomeric Aβ40 and Aβ42. Biophys J 104:2714–2724
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 278:42959–42965
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 18:1427–1429
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 72:248–254
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 24:743–750
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 32:1050–1060
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 37:11064–11077
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 269:5642–5648
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–S121
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 6:277–293
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 396:9–18
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 5:2336–2349
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 37:333–344
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 1784:2086–2092
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 267:169–184
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 421:554–560
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 14:1753–1759
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 126:1992–2005
Kollipara L, Zahedi RP (2013) Protein carbamylation: in vivo modification or in vitro artefact? Proteomics 13:941–944
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 55:5445–5451
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 1768:3135–3144
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 1768:2400–2408
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 40:183–189
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 17:1241–1248
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 79:16–24
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 154:1257–1268
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 102:17342–17347
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 5:75–86
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 100:722–730
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 14:788–792
Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747
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 105:18349–18354
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 99:16742–16747
Petkova AT, Yau WM, Tycko R (2006) Experimental constraints on quaternary structure in Alzheimer’s β-amyloid fibrils. Biochemistry 45:498–512
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:e20289
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 425:3338–3359
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:e73
Sani MA, Gehman JD, Separovic F (2011) Lipid matrix plays a role in Abeta fibril kinetics and morphology. FEBS Lett 585:749–754
Satakarni M, Curtis R (2011) Production of recombinant peptides as fusions with SUMO. Protein Expr Purif 78:113–119
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 103:702–710
Selkoe DJ (2012) Preventing Alzheimer’s disease. Science 337:1488–1492
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 405:570–583
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 359:801–805
Sisodia SS (1992) β-Amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci USA 89:6075–6079
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 233:293–298
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 44:787–796
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 7:257–267
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 411:312–316
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 59:687–696
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 276:1266–1281
Watson AA, Fairlie DP, Craik DJ (1998) Solution structure of methionine-oxidized amyloid β-peptide (1–40). Does oxidation affect conformational switching? Biochemistry 37:12700–12706
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–S293
Weber DK, Gehman JD, Separovic F, Sani MA (2012) Copper modulation of amyloid beta 42 interactions with model membranes. Aust J Chem 65:472–479
Williamson MP, Suzuki Y, Bourne NT, Asakura T (2006) Binding of amyloid β-peptide to ganglioside micelles is dependent on histidine-13. Biochem J 397:483–490
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 6:135–140
Yan Y, McCallum SA, Wang C (2008) M35 oxidation induces Aβ40-like structural and dynamical changes in Aβ42. J Am Chem Soc 130:5394–5395
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Weber, D.K., Sani, MA. & Gehman, J.D. A routine method for cloning, expressing and purifying Aβ(1–42) for structural NMR studies. Amino Acids 46, 2415–2426 (2014). https://doi.org/10.1007/s00726-014-1796-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-014-1796-x