单细菌水平蛋白质相互作用的高通量定量检测新方法
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- 英文论文题名：High-Throughput Single-cell Study of Protein-Protein Interactions
- 论文作者：吴丽娜 ; 汪旭 ; 张健强 ; 黄天逊 ; 颜晓梅
- 英文论文作者：Lina Wu ; Xu Wang ; Jianqiang Zhang ; Tianxun Huang ; Xiaomei Yan ; Department of Chemical Biology ; College of Chemistry and Chemical Engineering ; The Key Laboratory for Chemical Biology of Fujian Province ; The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation ; Xiamen
- 年：2016
- 作者机构：厦门大学化学化工学院化学生物学系化学生物学福建省重点实验室谱学分析与仪器教育部重点实验室;
- 论文关键词：细菌双杂交 ; 单个细菌水平 ; 流式检测技术 ; 蛋白质相互作用
- 会议召开时间：2016-07-01
- 会议录名称：中国化学会第30届学术年会摘要集-第二十八分会：化学生物学
- 语种：中文
- 分类号：Q51
- 学会代码：ZGHY
- 会议名称：中国化学会第30届学术年会-第二十八分会 ; 化学生物学
- 会议地点：中国辽宁大连
- 主办单位：中国化学会
- 学会名称：中国化学会
- 页数：1
- 文件大小：189k
- 原文格式：D
- 会议级别：全国
摘要
蛋白一蛋白相互作用研究是理解生命活动的基础,细菌双杂交系统已被广泛应用于蛋白质相互作用研究。单个细菌水平的蛋白一蛋白相互作用研究有助于更准确地考察蛋白在活细胞中的相互作用状况,揭示传统生物化学检测手段中因集权平均而被掩盖的个体差异。本研究通过流式检测技术与细菌双杂交系统的有机结合,发展灵敏、快速、高分辨、高通量的蛋白一蛋白相互作用定量研究新方法。利用流式分析技术,结合双色免疫荧光染色法,我们在单细菌水平对蛋白相互作用强弱(报告基因)和相互作用蛋白自身表达进行同时定量检测。提出相对报告基因表达量(单位相互作用蛋白所产生的报告基因表达量),并将其应用到不同相互作用强度蛋白对的检测和区分,建立了一种快速检测蛋白相互作用强度的新方法。
Here we report a high-throughput and quantitative in vivo method for the measurement of protein interactions at the single-cell level using bacterial adenylate cyclase two-hybrid system(BACTH).A multicolor flow cytometer was used to analyze the expression levels of the interacting proteins and to correlate with the expression ofβ-galactosidase(β-gal) reporter protein through dual immunofluorescence staining.The concept of relative]reporter protein expression(RRPE),defined as the normalized β-gal expression to that of the interacting protein,was introduced for affinity measure of the protein pair.The as-developed RRPE-BACTH method not only allows rapid in vivo detection of protein interaction in single cells but also provides relative affinity of the protein interaction.Due to the easy affinity ranking nature,RRPE-BACTH provides a quantitative screening platform for protein-protein interaction study.
Here we report a high-throughput and quantitative in vivo method for the measurement of protein interactions at the single-cell level using bacterial adenylate cyclase two-hybrid system(BACTH).A multicolor flow cytometer was used to analyze the expression levels of the interacting proteins and to correlate with the expression ofβ-galactosidase(β-gal) reporter protein through dual immunofluorescence staining.The concept of relative]reporter protein expression(RRPE),defined as the normalized β-gal expression to that of the interacting protein,was introduced for affinity measure of the protein pair.The as-developed RRPE-BACTH method not only allows rapid in vivo detection of protein interaction in single cells but also provides relative affinity of the protein interaction.Due to the easy affinity ranking nature,RRPE-BACTH provides a quantitative screening platform for protein-protein interaction study.
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