Recombinant Production and Purification of Aβ40 Variants

Commercial E. coli strain BL21 (DE3) cells (Agilent) were transformed with a pETSAC plasmid containing the sequence for Aβ40 [54] (link), and bacteria were grown in minimal medium for ¹³C, ¹⁵N-labeled samples [37] (link) or LB media for unlabeled samples [54] (link). Cultures were grown and Aβ40 purified as described previously [37] (link), [49] . An identical procedure was used for purification of both labeled and unlabeled samples. The resulting Aβ40 protein contains an additional N-terminal methionine residue that has no effect on the fibrillation of Aβ40 or the morphology of fibrils formed [54] (link). Final protein concentrations were estimated from UV absorption in 7 M guanidinium chloride at 280 nm using an extinction coefficient of 1490 M^− 1 cm^− 1. Site-directed mutagenesis was performed using a Q5 kit (NEB) to create each variant of Aβ40. Substitutions were verified by plasmid sequencing (Beckman-Coulter Genomics). All variants were expressed and purified as described for the WT sequence.