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Protein Expr Purif. 2012 Jan;81(1):11-17. doi: 10.1016/j.pep.2011.08.006. Epub 2011 Aug 31.

Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

Author information

1
Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, United States.
2
Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, United States. Electronic address: wangc5@rpi.edu.

Abstract

Alzheimer's disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid β peptide (Aβ). Aβ aggregation produces toxic oligomers and fibrils, causing neuronal dysfunction and memory loss. Aβ is generated from two sequential proteolytic cleavages of a membrane protein, amyloid precursor protein (APP), by β- and γ-secretases. The transmembrane (TM) domain of APP, APPTM, is the substrate of γ-secretase for Aβ production. The interaction between APPTM and γ-secretase determines the production of different species of Aβ. Although numerous experimental and theoretical studies of APPTM structure exist, experimental 3D structure of APPTM has not been obtained at atomic resolution. Using the pETM41 vector, we successfully expressed an MBP-APPTM fusion protein. By combining Ni-NTA chromatography, TEV protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically-labeled APPTM for NMR studies. The reconstitution of APPTM into micelles yielded high quality 2D (15)N-(1)H HSQC spectra. This reliable method for APPTM expression and purification lays a good foundation for future structural studies of APPTM using NMR.

PMID:
21907289
DOI:
10.1016/j.pep.2011.08.006
[Indexed for MEDLINE]

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