Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay.
Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay.
复制标题
使用Toxgreen（Toxgreen）（Toxcat分析的高通量变体）筛选除法体蛋白质中的跨膜关联。
DOI：
10.1016/j.bbamem.2016.07.008
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发表时间：
2016-11
期刊：
影响因子：
--
通讯作者：
Senes A
中科院分区：
文献类型：
--
作者：
Armstrong CR;Senes A
TOXCAT是一种广泛用于研究大肠杆菌内膜内跨膜螺旋相互作用的遗传测定方法。TOXCAT是基于一种融合构建体，将感兴趣的跨膜结构域与来自霍乱弧菌ToxR蛋白的细胞质dna结合结构域连接起来。跨膜结构域驱动的相互作用导致ToxR结构域二聚化，进而激活报告基因氯霉素乙酰转移酶（chloramphenicol acetyl transferase， CAT）的表达。CAT的定量被用来衡量跨膜结构域的自结合能力。由于CAT的定量比较费力，我们开发了一种高通量的变体TOXGREEN，基于超折叠GFP的表达和在未处理的细胞培养中直接检测荧光。对TOXCAT和TOXGREEN的仔细对比表明，这两种方法在LB和最小介质中都具有相当的响应、动态范围、灵敏度和内在变异性。大大增强的工作流程使TOXGREEN更具可扩展性，是筛选的理想选择，因为可以在96个孔板中快速评估数百个结构。即使是小规模的调查，TOXGREEN也能显著减少与程序相关的时间、人工和成本。我们通过对属于11种细菌的分裂体（FtsL, FtsB, FtsQ, FtsI, FtsN， ZipA和EzrA）的双位蛋白跨膜结构域的自我关联进行了大量筛选，证明了该方法的适用性。该分析证实了先前报道的FtsB自结合的倾向，并表明ZipA、EzrA和FtsN的跨膜结构域也可能发生寡聚。
TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.
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