%0 Journal Article
%T Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid beta 1-42
%J Methods and Protocols | An Open Access Journal from MDPI
%D 2019
%R https://doi.org/10.3390/mps2020048
%X Amyloid plaques found in the brains of Alzheimer¡¯s disease patients primarily consists of amyloid beta 1-42 (A¦Â42). Commercially, A¦Â42 is synthesized using high-throughput peptide synthesizers resulting in the presence of impurities and the racemization of amino acids that affects its aggregation properties. Furthermore, the repeated purchase of even a small quantity (~1 mg) of commercial A¦Â42 can be expensive for academic researchers. Here, we describe a detailed methodology for robust expression of recombinant human A¦Â(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli using standard molecular biology techniques with refined and rapid one-step analytical purification techniques. The peptide is isolated and purified from transformed cells using an optimized reverse-phase high-performance liquid chromatography (HPLC) protocol with commonly available C18 columns, yielding high amounts of peptide (~15¨C20 mg per 1 L culture) within a short period of time. The recombinant human A¦Â(M1-42) forms characteristic aggregates similar to synthetic A¦Â42 aggregates as verified by western blotting and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique produces pure recombinant human A¦Â(M1-42) that may be used to synthesize chemical probes and in several downstream in vitro and in vivo assays to facilitate Alzheimer¡¯s disease research. View Full-Tex
%U https://www.mdpi.com/2409-9279/2/2/48