文章基本信息

标题：Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells
本地全文：下载
作者：Miaomiao Xue ; Junjie Hou ; Linlin Wang 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2017
卷号：7
期号：1
DOI：10.1038/s41598-017-12365-9
语种：English
出版社：Springer Nature
摘要：Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.