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

doi:10.5246/jcps.2019.08.053

Journal of Chinese Pharmaceutical Sciences ›› 2019, Vol. 28 ›› Issue (8): 556-560.DOI: 10.5246/jcps.2019.08.053

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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).

Abstract

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

CLC Number:

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.

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

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