Cysteine misincorporation in bacterially expressed human alpha-synuclein.
Cysteine misincorporation in bacterially expressed human alpha-synuclein.
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发表时间：
2006
期刊：
影响因子：
3.5
通讯作者：
Masami Masuda;N. Dohmae;T. Nonaka;Takayuki Oikawa;S. Hisanaga;M. Goedert;M. Hasegawa
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文献类型：
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作者：
Masami Masuda;N. Dohmae;T. Nonaka;Takayuki Oikawa;S. Hisanaga;M. Goedert;M. Hasegawa
细菌表达的人α-突触核蛋白(α-syn)已广泛应用于结构和功能研究。在这里，我们发现在大肠杆菌中表达的人α-syn大约有20%是错误翻译的，并且半胱氨酸残基被掺入在第136位，而不是Tyr。密码子136(TAC到TAT)的定点突变导致缺乏Cys的α-syn的表达。虽然野生型(Y136-TAC和Y136-TAT)和突变型(C136-TGC)α-syn有相似的聚集成丝的倾向，但误掺入会增加二聚体α-syn的水平。为了避免潜在的伪影，我们建议使用Y136-TAT结构来表达人α-syn。
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.