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

作者：Weber Daniel K; Sani Marc Antoine; Gehman John D^*

来源：Amino Acids, 2014, 46(10): 2415-2426.

DOI：10.1007/s00726-014-1796-x

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

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-beta peptide (A beta) 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 A beta. Furthermore, the notorious propensity of A beta 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 N-15 and (CN)-C-13-N-15 A beta(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.

• 出版日期2014-10