Designed Phosphoprotein Recognition in Escherichia coli

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• 作者：Nicholas Sawyer ; Brandon M. Gassaway ; Adrian D. Haimovich ; Farren J. Isaacs ; Jesse Rinehart ; Lynne Regan
• 刊名：ACS Chemical Biology
• 出版年：2014
• 出版时间：November 21, 2014
• 年：2014
• 卷：9
• 期：11
• 页码：2502-2507
• 全文大小：530K
• ISSN：1554-8937

文摘

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide鈥損rotein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide鈥損rotein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide鈥損rotein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.