Interaction-dependent native chemical ligation and protein trans-splicing (IDNCL-PTS) for detection and visualization of ligand-protein interactions
Interaction-dependent native chemical ligation and protein trans-splicing (IDNCL-PTS) for detection and visualization of ligand-protein interactions
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相互作用依赖性天然化学连接和蛋白质转拼 (IDNCL-PTS)，用于配体-蛋白质相互作用的检测和可视化
DOI：
10.1002/slct.201600443
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发表时间：
2016
期刊：
影响因子：
2.1
通讯作者：
A. Saito
中科院分区：
文献类型：
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作者：
T. Takahashi;A. Saito
基于相互作用依赖的天然化学连接和蛋白质反式剪接（IDNCL‐PTS），我们构建了一个检测系统，通过特定的酶活性来可视化配体与蛋白质的相互作用。我们设计了β -半乳糖苷酶（β gal），能够通过蛋白质反式剪接（PTS）产生酶活性，使用来自synechocystissp的分裂蛋白。PCC6803 (Ssp) dna。两个短肽活性标签，每个都有βGal（24-34）或DnaB（1-11）的序列，被结合到配体和靶蛋白中，配体-蛋白的相互作用通过邻近效应促进肽标签之间的连接。结合IDNCL和PTS，通过βGal活性清晰地检测了碳水化合物与麦芽糖结合蛋白（MBP）的相互作用，以及磷脂与肽基脯氨酸顺/反异构酶NIMA相互作用1 （Pin1）的相互作用。有趣的是，磷脂肽- Pin1相互作用在粗质周提取物中直接可见。
We constructed a detection system to visualize ligand‐protein interactions by specific enzyme activity based on interaction‐dependent native chemical ligation and protein trans‐splicing (IDNCL‐PTS). We engineered β‐galactosidase (βGal) capable of generating enzyme activity through protein trans‐splicing (PTS) using a split intein derived fromSynechocystissp. PCC6803 (Ssp) DnaB. Two short peptide reactive tags, each having the sequence of βGal(24–34) or DnaB(1–11), were incorporated into a ligand and a target protein, and the ligand‐protein interaction promoted the ligation between the peptide tags by a proximity effect. Combining IDNCL and PTS, interactions of carbohydrates with maltose binding protein (MBP) and phosphopeptides with peptidyl‐prolys cis/trans isomerase NIMA‐interacting 1 (Pin1) were clearly detected by βGal activity. Interestingly, phosphopeptide‐Pin1 interactions were visualized directly in crude periplasmic extracts.