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Cysteine misincorporation in bacterially expressed human α‐synuclein
Author(s) -
Masuda Masami,
Dohmae Naoshi,
aka Takashi,
Oikawa Takayuki,
Hisanaga Shin-ichi,
Goedert Michel,
Hasegawa Masato
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.02.032
Subject(s) - cysteine , escherichia coli , mutant , mutagenesis , alpha synuclein , site directed mutagenesis , biochemistry , chemistry , alpha (finance) , biology , amino acid , mutation , microbiology and biotechnology , genetics , gene , enzyme , medicine , construct validity , nursing , disease , pathology , parkinson's disease , patient satisfaction
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.