基于红色荧光蛋白的酵母双分子荧光互补技术的建立

yEmRFP-based bimolecular fluorescence complementary technology in yeast

目的在酵母细胞中建立基于酵母增强型红色荧光蛋白(yEmRFP)的双分子荧光互补技术,用于直观地检测蛋白质相互作用。方法利用分子克隆技术将yEmRFP的N端片段(1~159 aa,RN159)和C端片段(160~236 aa,RC160)分别与转录因子Jun和Fos的碱性亮氨酸拉链结构域(bZIP)—bJun和bFos或其缺失突变体bFosΔZip相融合,构建重组质粒,其中bFosΔZip与bJun不存在相互作用,可作为阴性对照组,将重组质粒转化酵母细胞后,通过营养缺陷培养基筛选转化成功的酵母菌落,利用荧光显微镜和流式细胞仪检测酵母中重组yEmRFP蛋白质的荧光信号,判定该技术是否可用于检测蛋白质相互作用。结果成功构建了RN159和RC160与bJun和bFos或bFosΔZip相融合的重组质粒。酵母表达体系中bJun和bFos的相互作用使yEmRFP重新组装为完整荧光蛋白质,通过荧光显微镜检测到强的红色荧光信号,利用流式细胞仪藻红蛋白(PE)通道计数20000个酵母细胞,其中约有25%酵母细胞具有红色荧光;而阴性对照组未检测到荧光信号,同样在流式细胞仪PE通道中未检测到明显的红色荧光信号,表明此体系无自发激活现象。结论成功开发了基于yEmRFP的酵母双分子荧光互补技术,可用于直观、快速检测和筛选蛋白质相互作用。

Objective To establish a bimolecular fluorescence complementary technology based on a red fluorescent protein,yEmRFP,for detection of protein-protein interactions in yeast cells.Methods The N-terminal fragment(1-159 aa,RN159)and C-terminal fragment(160-236 aa,RC160)of yEmRFP were fused with bJun and bFos or bFosΔZip,respectively,to construct recombinant plasmids via molecular cloning technology.Among them,bJun and bFos were the basic leucine zipper domains(bZIP)of transcription factors Jun and Fos,and there was no interaction between bFosΔZip and bJun,which could be used as a negative control.After the recombinant plasmids were transformed into yeast cells,the transformed yeast colonies were screened in auxotrophic medium,and the fluorescence signals of recombinant yEmRFP protein in yeast were detected by fluorescence microscopy and flow cytometry.Thus,it was confirmed that this method was effective for investigating protein-protein interactions.Results The recombinant plasmids of RN159 and RC160 fused with bJun and bFos or bFosΔZip were constructed.The interactions between bJun and bFos reassembled yEmRFP into complete fluorescent protein in yeast cells and strong red fluorescence signals were detected by fluorescence microscopy.About 25%of the 20000 yeast cells counted by the phycoerythrin(PE)channel of flow cytometry had red fluorescence signals.No fluorescence signal was detected in the negative control group,and no red fluorescentce signal was clearly detected in the same PE channel of the flow cytometry,thus indicating that this system had no self-activation.Conclusion In this study,a yEmRFP-based yeast bimolecular fluorescence complementation technology has been developed,which can be used for direct and rapid detection and screening of protein-protein interactions.