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

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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.

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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

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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.

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The authors declare no conflict of interest.

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Correspondence to John D. Gehman.

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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

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  • DOI: https://doi.org/10.1007/s00726-014-1796-x

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