Using an AraC-based three-hybrid system to detect biocatalysts in vivo

被引：40

作者：

Firestine, SM ^{[1
]}

Salinas, F ^{[1
]}

Nixon, AE ^{[1
]}

Baker, SJ ^{[1
]}

Benkovic, SJ ^{[1
]}

机构：

[1] Penn State Univ, Dept Chem, University Pk, PA 16802 USA

来源：

NATURE BIOTECHNOLOGY | 2000年 / 18卷 / 05期

基金：

美国国家卫生研究院;

关键词：

molecular evolution; screening/selection methods; scytalone dehydratase; three-hybrid systems; artificial transcription system;

D O I：

10.1038/75414

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Recent methods to create large libraries of proteins have greatly advanced the discovery of proteins with novel functions. However, one limitation in the discovery of new biocatalysts is the screening or selection methods employed to find enzymes from these libraries. We have developed a potentially general method termed QUEST (QUerying for EnzymeS using the Three-hybrid system), which allows the construction of an easily screened or selected phenotype for, in theory, any type of enzymatic reaction. The method couples the in vivo concentration of an enzyme's substrate to changes in the transcriptional level of a reporter operon. Using the arabinose operon activator AraC, we constructed a system capable of detecting the fungal enzyme scytalone dehydratase (SD) in bacteria, and demonstrated its sensitivity and usefulness in library screening.

引用

页码：544 / 547

页数：4

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