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Alzheimer´s Disease-associated Aβ42 Peptide: Expression and Purification for NMR Structural Studies

[ Vol. 11 , Issue. 1 ]

Author(s):

Montserrat Serra-Batiste, Raquel Garcia-Castellanos, Marti Ninot-Pedrosa, Bernat Serra-Vidal, Nicholas Simon Berrow and Natalia Carulla   Pages 50 - 62 ( 13 )

Abstract:


Background: The aggregation of the amyloid-beta peptide (Aβ) in the brain is strongly associated with Alzheimer´s disease (AD). However, the heterogeneous and transient nature of this process has prevented identification of the exact molecular form of Aβ responsible for the neurotoxicity observed in this disease. Therefore, characterizing Aβ aggregation is of utmost importance in the field of AD. Nuclear magnetic resonance spectroscopy (NMR) is a technique that holds great potential to achieve this goal. However, it requires the use of specific labels introduced through recombinant expression of Aβ.

Objective: In this paper, we report on a straightforward expression and purification protocol to obtain [U-15N] and [U-2H,13C,15N] Aβ42.

Method: Aβ42 is expressed fused to Small Ubiquitin-like Modifier (SUMO) protein, which prevents Aβ42 aggregation.

Results: The solubilizing capacity of SUMO has allowed us to design a purification protocol involving immobilized metal affinity chromatography (IMAC), a desalting step, and two size exclusion chromatography (SEC) purifications.

Conclusion: This approach, which does not require the use of costly and time-consuming reversed phase high performance liquid chromatography (RP-HPLC), offers a much straightforward strategy to those previously described to obtain [U-15N] Aβ42 and it is the first protocol through which to achieve [U-2H,13C,15N] Aβ42. The peptides obtained are of high purity and have the required isotope enrichment to support NMR-based structural studies.

Keywords:

Alzheimer's disease, Aβ42 peptide, isotope labeling, NMR, soluble expression, SUMO.

Affiliation:

Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028 Barcelona, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028 Barcelona, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028 Barcelona, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028 Barcelona, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028 Barcelona, 2 Rue Robert Escarpit, 33607 Pessac

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