基于大肠杆菌CAP蛋白的检测cAMP的新型荧光探针

doi:10.5246/jcps.2019.08.053

中国药学(英文版) ›› 2019, Vol. 28 ›› Issue (8): 556-560.DOI: 10.5246/jcps.2019.08.053

基于大肠杆菌CAP蛋白的检测cAMP的新型荧光探针

张嘉远, 宋雨欣, 王楚宸, 王晶^*

北京大学医学部药学院化学生物学系; 天然药物及仿生药物国家重点实验室, 北京 100191

收稿日期:2019-05-06 修回日期:2019-06-12 出版日期:2019-09-02 发布日期:2019-06-15
通讯作者: Tel.: +86-10-82801563, E-mail: wangjingsioc@pku.edu.cn
基金资助:
National Basic Research Foundation of China (Grant No. 2017YFA0505202), the National Natural Science Foundation of China (Grant No. 91853107).

A novel fluorescent sensor for cAMP based on E. coli CAP protein

Jiayuan Zhang, Yuxin Song, Chuchen Wang, Jing Wang^*

State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2019-05-06 Revised:2019-06-12 Online:2019-09-02 Published:2019-06-15
Contact: Tel.: +86-10-82801563, E-mail: wangjingsioc@pku.edu.cn
Supported by:
National Basic Research Foundation of China (Grant No. 2017YFA0505202), the National Natural Science Foundation of China (Grant No. 91853107).

摘要/Abstract

摘要：

cAMP是细胞内一种重要的信号分子,作为第二信使调控包括糖代谢、糖原代谢和脂质代谢在内的一系列生理过程。在本文中我们报道一个新颖的检测细胞内cAMP的基因编码荧光探针,这一探针是由大肠杆菌CAP蛋白和cpYFP荧光蛋白组成的融合蛋白,针对cAMP的结合可以产生一倍的荧光信号响应。进一步的表征实验表明,这一探针对cAMP具有很强的结合能力,并且能够实现快速地荧光响应。这一探针的开发将会为目前已有的检测cAMP的手段提供有益的补充。

关键词: 环腺苷酸, 荧光探针, 蛋白质工程, 信号转导

Abstract:

cAMP is an important second messenger that is capable of controlling a wide array of cellular processes, including glycogen, sugar and lipid metabolism. Here we report the design and construction of a novel genetically encoded fluorescent sensor for cAMP. The sensor was realized by fusing E. coli CAP protein with cpYFP, and displayed a one-fold fluorescence change towards cAMP binding. Further characterization assays demonstrated that the sensor had high affinity for cAMP and fast response kinetics. The development of our sensor could be a useful supplement to existing methods for cAMP detection.

Key words: cAMP, Fluorescent sensor, Protein engineering, Signal transduction

中图分类号:

R966

Supporting:

Figure S1. Cloning and purification of CAP-cpFP fusions. (A) PCR amplification of CAP gene sequence (653 bp) from E. coli strain BL21 genomic DNA. (B) Validation of CAP/pET-28a plasmid through colony PCR. (C) Purity validation of CAP-cpFP fusion proteins (54.8 kDa) by SDS-PAGE followed by Comassie Brilliant Blue staining.

Figure S2. Fluorescence response of CAP-cpFP fusions. (A-F) Fluorescence excitation and emission spectra of purified CAP-L137-cpYP (A), CAP-L137-cpGD (B), CAP-D138-cpYH (C), CAP-D138-cpYP (D), CAP-D138-cpYM (E) and CAP-D138-cpGD (F) in their apo states and after addition of 100 µM cAMP.

Figure S3. Gene and amino acid sequence of CAPY137. CAP, cpYH and linker regions are denoted in purple, yellow and black, respectively.

张嘉远, 宋雨欣, 王楚宸, 王晶*. 基于大肠杆菌CAP蛋白的检测cAMP的新型荧光探针[J]. 中国药学(英文版), 2019, 28(8): 556-560.

Jiayuan Zhang, Yuxin Song, Chuchen Wang, Jing Wang. A novel fluorescent sensor for cAMP based on E. coli CAP protein[J]. Journal of Chinese Pharmaceutical Sciences, 2019, 28(8): 556-560.

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基于大肠杆菌CAP蛋白的检测cAMP的新型荧光探针

A novel fluorescent sensor for cAMP based on E. coli CAP protein

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