Cysteine misincorporation in bacterially expressed human alpha-synuclein.
Bacterially expressed human alpha-synuclein (alpha-syn) has been widely used in structural and functional studies. Here we show that approximately 20% of human alpha-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 alpha-syn lacking Cys. Although wild-type (Y136-TAC and Y136-TAT) and mutant (C136-TGC) alpha-syn had similar propensities to assemble into filaments, the levels of dimeric alpha-syn were increased by misincorporation. To avoid potential artefacts, we recommend use of the Y136-TAT construct for the expression of human alpha-syn.
Masuda
Masami
M
Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku 156-8585, Tokyo, Japan.
Dohmae
Naoshi
N
Nonaka
Takashi
T
Oikawa
Takayuki
T
Hisanaga
Shin-ichi
S
Goedert
Michel
M
Hasegawa
Masato
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular / Q000379:methods*; D003545:Cysteine / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D051844:alpha-Synuclein / Q000235:genetics*
D051844:alpha-Synuclein; D003545:Cysteine