De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters

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• 作者：Yu Liu ; Xin Zhang ; Yun Lei Tan ; Gira Bhabha ; Damian C. Ekiert ; Yakov Kipnis ; Sinisa Bjelic ; David Baker ; Jeffery W. Kelly
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：September 24, 2014
• 年：2014
• 卷：136
• 期：38
• 页码：13102-13105
• 全文大小：324K
• ISSN：1520-5126

文摘

Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme鈥搒mall-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.