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- W2896640651 abstract "To understand Alzheimer's Disease, researchers need to obtain large quantities of pure β-Amyloid (Aβ) and related peptides, e.g., transmembrane domain of β-APP (TM-β-APP). Aβ and TM-β-APP are only sparingly water soluble in the absence of detergents and/or denaturants, but are usually expressed as fusion proteins with folded partners such as GST. In addition, cleavage of fusion proteins usually requires (folded) enzymes e.g., Factor Xa. To address these issues, we developed a versatile new bacterial expression system for producing large quantities of Aβ, TM-β-APP and related proteins, that does not require a folded fusion partner or enzymes. After standard expression techniques, unlabeled or isotopically labeled Aβ or TM-β-APP peptides were purified (≥ 95% by SDS-PAGE, LC-MS and NMR) using Ni-affinity chromatography, HPLC and FPLC. Yields and purity were sufficient for 2D and 3D NMR spectroscopy. Additional studies included electron microscopy, atomic force microscopy and dynamic light scattering. The expression system used a His6-tag instead of a folded protein fusion partner, and in some cases, a chemical instead of enzymatic cleavage site between Aβ and fusion partner. Transformation was straightforward, and yields were high (∼1-3 mg/L in various culture media). Peptides were purified using the relative harsh conditions needed for RP-HPLC or other chromatographic modalities, e.g., SEC with SDS. We obtained high quality 2D and 3D NMR spectra of the products for backbone assignments by triple resonance techniques. We have described a versatile and efficient method for expressing Aβ and related peptides, presumably applicable to other fibrillizing peptides. Uniquely, it does not require a folded fusion tag or enzyme for processing the lysate. Aβ and TM-β-APP strongly self-associate, so their purification generally requires using denaturants and/or detergents. Our expression system allows the use of such relatively harsh conditions, and thus yields large quantities of pure unlabeled or isotopically labeled β-amyloid family peptides." @default.
- W2896640651 created "2018-10-26" @default.
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- W2896640651 date "2018-07-01" @default.
- W2896640651 modified "2023-10-16" @default.
- W2896640651 title "P2‐221: A VERSATILE EXPRESSION AND PURIFICATION METHOD FOR ISOTOPICALLY LABELED β‐AMYLOID FAMILY PEPTIDES" @default.
- W2896640651 doi "https://doi.org/10.1016/j.jalz.2018.06.909" @default.
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