Cysteine misincorporation in bacterially expressed human α-synuclein

Bacterially expressed human α-synuclein (α-syn) has been widely used in structural and functional studies. Here we show that ∼20% of human α-syn expressed in Escherichia coli is mistranslated and that a Cys residue is incorporated at position 136 instead of a Tyr. Site-directed mutagenesis of codon 136 (TAC to TAT) resulted in the expression of α-syn lacking Cys. Although wild-type (Y136-TAC and Y136-TAT) and mutant (C136-TGC) α-syn had similar propensities to assemble into filaments, the levels of dimeric α-syn were increased by misincorporation. To avoid potential artefacts, we recommend use of the Y136-TAT construct for the expression of human α-syn.