Abstract
The Alzheimer’s disease related peptide, Amyloid-beta (Aβ)1–40 and 1–42, has proven difficult to be purified as a recombinant monomeric protein due its expression in E. coli leading to the formation of insoluble inclusion bodies and its tendency to quickly form insoluble aggregates. A vast array of methods have been used so far, yet many have pitfalls, such as the use of tags for ease of Aβ isolation, the formation of Aβ multimers within the time frame of extraction, or the need to reconstitute Aβ from a freeze–dried state. Here, we present a rapid protocol to produce highly pure and monomeric recombinant Aβ using a one-step ion exchange purification method and to label the peptide using a maleimide dye. The washing, solubilization, and purification steps take only 3 h. We also present a protocol for the isolation of Aβ-mCherry from mammalian cells.
Highlights
The presence of Amyloid-beta (Aβ) plaques and τ angles in neurons are hallmarks of Alzheimer’s disease, great research effort is put toward understanding how these initially soluble proteins misfold and contribute to pathology
We present a protocol for the isolation of AβM(E22G)-mCherry from HEK293 cells again cells by sonication and centrifuged at 10,000g
Aβ is commonly degraded or altered by post-translational modification; the presence of species of differing molecular weights of AβM(E22G)-mCherry may be reflective of different truncations and modifications that occur within a cellular environment.[22]
Summary
The presence of Amyloid-beta (Aβ) plaques and τ angles in neurons are hallmarks of Alzheimer’s disease, great research effort is put toward understanding how these initially soluble proteins misfold and contribute to pathology. Large quantities of protein are required, and while purification protocols for τ proteins are fairly well established, those for Aβ, in its isoforms of 1-39/43 and its mutant variants, are very heterogeneous and lead to variable products.[1]. There is often variation in the resulting sample due the use of different solvents for reconstitution, which can influence the initial structure.[2] Differences in disaggregation protocols can introduce heterogeneous structures in the starting peptide, such as the use of hexafluoroisopropanol (HFIP) vs ammonium hydroxide. HFIP is widely used to disaggregate Aβ, yet it can lead to formation of oligomeric structures, as the peptide is brought to neutral pH via its isoelectric point.[3] Ammonium hydroxide disaggregation has been shown to produce fewer oligomers and a more homogeneous solution compared to a HFIP prepared Aβ solution.
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