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

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.