一种用于氨酰-tRNA合成酶快速进化的质粒选择系统。
One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases.
作者信息
Melançon Charles E, Schultz Peter G
机构信息
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
出版信息
Bioorg Med Chem Lett. 2009 Jul 15;19(14):3845-7. doi: 10.1016/j.bmcl.2009.04.007. Epub 2009 Apr 9.
Abstract
We have developed a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specificities in Escherichia coli. This system utilizes an amber stop codon containing chloramphenicol acetyltransferase/uracil phosphoribosyltransferase fusion gene. We demonstrate the utility of the system by identifying a variant of the Methanococcus jannaschii tyrosyl synthetase from a library of 10(9) variants that selectively incorporates para-iodophenylalanine in response to an amber stop codon.
摘要
我们开发了一种快速、直接的单质粒双阳性/阴性选择系统，用于在大肠杆菌中对特异性改变的氨酰基-tRNA合成酶进行进化。该系统利用了一个含有氯霉素乙酰转移酶/尿嘧啶磷酸核糖基转移酶融合基因的琥珀色终止密码子。我们通过从一个包含10^9个变体的文库中鉴定出詹氏甲烷球菌酪氨酰合成酶的一个变体，证明了该系统的实用性，该变体可响应琥珀色终止密码子选择性地掺入对碘苯丙氨酸。
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