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High-yield expression and purification of the transmembrane region of ion channel-forming amyloid-β protein for NMR structural studies

Paper ID Volume ID Publish Year Pages File Format Full-Text
35671 45101 2010 7 PDF Available
Title
High-yield expression and purification of the transmembrane region of ion channel-forming amyloid-β protein for NMR structural studies
Abstract

Numerous studies of APP in the literature to date have focused on its processing at the plasma membrane, its membrane-bound oligomeric state, and the calcium-permeable ion channel formation of non-fibrillar Aβ in the cell membrane. Despite these studies, little is known structurally about the transmembrane region of APP beyond a theoretical model. This is due to challenges in the expression, purification, and sample preparation of eukaryotic membrane proteins. To determine the three-dimensional structures of the intact transmembrane domain from human APP (hAPP-TM) and to elucidate the structure and formation mechanisms of the hAPP channel, the isotopically labeled protein must be produced and purified in sufficient quantity. Here, we describe a procedure whereby the hAPP-TM peptide, comprising residues 692–723 of hAPP, was successfully expressed and purified sufficiently to perform NMR analysis. To increase expression levels of the target protein, we designed a construct containing two tandem repeats of the target gene. The fusion protein was expressed in the form of inclusion bodies, purified on immobilized nickel affinity chromatography, and chemically cleaved by cyanogen bromide. Final purification of hAPP-TM was achieved by preparative reversed-phase high performance liquid chromatography (HPLC). The final yields of purified hAPP-TM were around 5 mg/l of M9 minimal media.

Keywords
Amyloid channel; Integral membrane protein; Transmembrane protein; Multimeric coding sequence; Alzheimer's disease; NMR
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High-yield expression and purification of the transmembrane region of ion channel-forming amyloid-β protein for NMR structural studies
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 45, Issue 5, May 2010, Pages 682–688
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us