GFP报告因子结合流式细胞术的蛋白-蛋白相互作用单细菌水平分析
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摘要
在基于腺苷酸环化酶功能重构的细菌双杂交(BACTH)系统中引入lac启动子控制的gfp基因作为蛋白质相互作用的报告基因,采用实验室自行研制的超高灵敏流式检测装置(HSFCM)建立了一种单细菌水平的、简单、快速、高通量的蛋白-蛋白相互作用分析方法.将分别携带有相互作用蛋白对编码基因和gfp报告基因的质粒共转化至报告菌株,利用HSFCM对单个细菌的散射光和荧光同时进行检测.结果表明蛋白-蛋白相互作用激活了gfp报告基因的表达,并存在双稳态现象:即一部分细菌的gfp报告基因被激活,细菌荧光信号强度明显高于阴性对照组,而另一部分细菌的gfp报告基因未被激活,细菌荧光信号分布与阴性对照组重叠.利用HSFCM在单细菌水平对荧光信号强度和表达GFP报告因子的细菌比例进行分析,能有效地区分表达GFP报告因子的阳性菌和报告基因未被激活表达的阴性菌,揭示了细菌个体蛋白的异质性表达.这将为相互作用蛋白对的检测及筛选提供一种快速的分析方法.
Alac controlled gfpgene 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 analysis of protein-protein interactions.In this method,Escherichia coli BTH101 was co-transformed with two compatible plasmids containing the interacting protein pair and the gfpgene,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 proteinprotein 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 interacting protein pairs.
Alac controlled gfpgene 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 analysis of protein-protein interactions.In this method,Escherichia coli BTH101 was co-transformed with two compatible plasmids containing the interacting protein pair and the gfpgene,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 proteinprotein 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 interacting protein pairs.
引文
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[6]GAULIARD E,OUELLETTE S P,RUEDEN K J,et al.Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis[J].Front Cell Infect Microbiol,2015,5:13.
[7]PASCHOS A,DEN HARTIGH A,SMITH M A,et al.An in vivo high-throughput screening approach targeting the typeⅣsecretion system component VirB8identified inhibitors of Brucella abortus 2308proliferation[J].Infect Immun,2011,79(3):1033-1043.
[8]ZOUED A,DURAND E,BRUNET Y R,et al.Priming and polymerization of a bacterial contractile tail structure[J].Nature,2016,531(7592):59.
[9]TAO L,PENG L S,BERNTSSON R P,et al.Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors[J].Nat Commun,2017,8(1):53-63.
[10]VIDAL-AROCA F,GIANNATTASIO M,BRUNELLI E,et al.One-step high-throughput assay for quantitative detection of beta-galactosidase activity in intact gramnegative bacteria,yeast,and mammalian cells[J].Biotechniques,2006,40(4):433-438.
[11]TANIGUCHI Y,CHOI P J,LI G W,et al.Quantifying E.coli proteome and transcriptome with single-molecule sensitivity in single cells[J].Science,2010,329(5991):533-538.
[12]NOLAN J P,YANG L.The flow of cytometry into systems biology[J].Brief Funct Genomic Proteomic,2007,6(2):81-90.
[13]ZHU S B,MA L,WANG S,et al.Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles[J].ACS Nano,2014,8(10):10998-11006.
[14]HE S B,HONG X Y,HUANG T X,et al.Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry[J].Methods Appl Fluoresc,2017,5(2):024002.
[15]WU L N,SONG Y Y,LUAN T,et al.Specific detection of live Escherichia coli O157:H7 using tetracysteinetagged PP01 bacteriophage[J].Biosens Bioelectron,2016,86:102-108.
[16]HUANG T X,ZHENG Y,YAN Y,et al.Probing minority population of antibiotic-resistant bacteria[J].Biosens Bioelectron,2016,80:323-330.
[17]YU M X,WU L N,HUANG T X,et al.Rapid detection and enumeration of total bacteria in drinking water and tea beverages by a laboratory-built high-sensitivity flow cytometer[J].Analytical Methods,2015,7(7):3072-3079.
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[19]WU L N,WANG X,ZHANG J Q,et al.Flow cytometric single-cell analysis for quantitative in vivo detection of protein-protein interactions via relative reporter protein expression measurement[J].Anal Chem,2017,89(5):2782-2789.
[20]YU D,BAIRD M A,ALLEN J R,et al.A naturally monomeric infrared fluorescent protein for protein labeling in vivo[J].Nat Methods,2015,12(8):763-765.
[21]GIBSON D G,YONG L,CHUANG R Y,et al.Enzymatic assembly of DNA molecules up to several hundred kilobases[J].Nat Methods,2009,6(5):343-345.
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