Establishment of tetracycline-regulated bimolecular fluorescence complementation assay to detect protein-protein interactions in Candida albicans

doi:10.1038/s41598-020-59891-7

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 期刊論文 > Item 310902500/20635

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题名:	Establishment of tetracycline-regulated bimolecular fluorescence complementation assay to detect protein-protein interactions in Candida albicans
作者:	Lai, W.-C. Sun, H.S. Shieh, J.-C.
关键词:	Candida albicans;Virulence;Human fungal
日期:	2020-12-01
上传时间:	2020-03-11T04:27:04Z (UTC)
出版者:	Scientific Reports
ISSN:	2045-2322
摘要:	To visualize protein-protein interactions in Candida albicans with the bimolecular fluorescence complementation (BiFC) approach, we created a Tet-on system with the plasmids pWTN1 and pWTN2. Both plasmids bear a hygromycin B-resistant marker (CaHygB) that is compatible with the original Tet-on plasmid pNIM1, which carries a nourseothricin-resistant marker (CaSAT1). By using GFPmut2 and mCherry as reporters, we found that the two complementary Tet-on plasmids act synergistically in C. albicans with doxycycline in a dose-dependent manner and that expression of the fusion proteins, CaCdc11-GFPmut2 and mCherry-CaCdc10, derived from this system, is septum targeted. Furthermore, to allow detection of protein-protein interactions with the reassembly of a split fluorescent protein, we incorporated mCherry into our system. We generated pWTN1-RN and pNIM1-RC, which express the N-terminus (amino acids 1–159) and C-terminus (amino acids 160–237) of mCherry, respectively. To verify BiFC with mCherry, we created the pWTN1-CDC42-RN (or pWTN1-RN-CDC42) and pNIM1-RC-RDI1 plasmids. C. albicans cells containing these plasmids treated with doxycycline co-expressed the N- and C-terminal fragments of mCherry either N-terminally or C-terminally fused with CaCdc42 and CaRdi1, respectively, and the CaCdc42-CaRdi1 interaction reconstituted a functional form of mCherry. The establishment of this Tet-on-based BiFC system in C. albicans should facilitate the exploration of protein-protein interactions under a variety of conditions. © 2020, The Author(s).
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/20635
關聯:	Scientific Reports, Volume 10, Issue 1, no. 2936
显示于类别:	[生化微生物免疫研究所] 期刊論文

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