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Construction of CFP-labeled Protein Library and Screening Calmodulin Binding Proteins by FRET Technology
Meng Fanli1,2, Guan Shan1, Liu Xiaojin1, Li Chaojun3*
1College of Life Sciences, Nanjing Normal University, Nanjing 210046, China; 2School of Basic Medical Sciences,Nanjing Medical University, Nanjing 210029, China; 3Model Animal Research Center (MARC) and the School of Medic
Abstract: Calmodulin (CaM)， the main receptor for intracellular Ca2+ signals， regulates the activity of its target proteins by interacting with them and plays an important role in the cell proliferation， differentiation， apoptosis， migration， etc. Fluorescence resonance energy transfer (FRET) technology is one of the mature methods for studying proteins interactions. By applying Cre-loxP site-specific recombination technology we constructed CFP-labeled library， cotransfected HEK293 cells with YFP-CaM plasmids， and used fluorescence resonance energy transfer (FRET) technology to detect the protein interaction. We picked up the cells which generated FRET and applied single-cell PCR detection. By sequencing the PCR products and comparing them with the database NCBI， we screened the unknown proteins which interacted with CaM. Taken together， we have found some CaM binding proteins through the construction of CFP-labeled protein library and applying the FRET technology. Our study provides the conditions for further study of CaM protein in physical environment.
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