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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

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.

Keywords

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

Abbreviations

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

Notes

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.

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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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