(厦门大学 化学化工学院,谱学分析与仪器教育部重点实验室,化学生物学福建省重点实验室,福建 厦门 361005)

蛋白-蛋白相互作用; BACTH系统; GFP报告因子; 单细菌水平; 超高灵敏流式检测技术

A GFP Reporter Protein Combined with Flow Cytometry for Analyzing Protein-protein Interactions at the Single-bacterium Level
SU Liuqin,ZHANG Jianqiang,HE Shengbin,WU Lina*,YAN Xiaomei

(Key Laboratory of Spectrochemical Analysis & Instrumentation,Ministry of Education,The Key Laboratory for Chemical Biology of Fujian Province,College of Chemistry and Chemical Engineering,Xiamen University,Xiamen 361005,China)

protein-protein interaction; BACTH system; GFP reporter protein; single-bacterium analysis; high sensitivity flow cytometry

DOI: 10.6043/j.issn.0438-0479.201803041



A lac controlled gfp gene was introduced into the bacterial adenylate cyclase two-hybrid(BACTH)system,and a laboratory-built high sensitivity flow cytometer(HSFCM)was used to establish a simple method for the rapid and high throughput analy-sis of protein-protein interactions.In this method,Escherichia coli BTH101 was co-transformed with two compatible plasmids containing the interacting protein pair and the gfp gene,respectively.The GFP expression of the bacteria was determined using the HSFCM through side scattering(SS)and fluorescence(FL)analysis.It was found that the expression of GFP was activated by the protein-protein interactions,and there exist an all-or-none phenomenon,i.e.,a fraction of the bacterial cells population highly expressed GFP(GFP+),whereas the remainder of the cells did not.The proportion of GFP+ population and the FL intensity were believed to be related to the association constant of the two proteins(Pal-TolB).Through the analysis of the FL intensity and proportion of bacteria expressing GFP reporter protein at the single-bacterium level,we can effectively discriminate the GFP+ bacteria from negative ones,and reveal the population heterogeneity.Hence,the flow cytometric assay may provide a powerful analytical tool for screening inte-racting protein pairs.