Analysis of Protein-Protein Interactions by Split Luciferase Complementation Assay

Current protocols in toxicology Pub Date : 2019-12-02 DOI:10.1002/cptx.90

Yuekun Lang, Zhong Li, Hongmin Li

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引用次数: 10

Abstract

Protein-protein interactions are important in human disease. Developing and refining tools to understand physical contacts between signaling proteins is crucial. This article describes a split luciferase complementation (SLC) method designed to discover inhibitors of protein-protein interaction. Different fusion proteins with split luciferase are constructed, expressed, and purified, and then assessed to determine the best pair that generates the strongest luminescence. SLC specificity and affinity are further confirmed. Step-by-step instructions are provided for performing these assays using the NS2B-NS3 interaction as an example. NS2B is an essential cofactor for flaviviral NS3 protease function. Advantages and disadvantages of these assays are further discussed. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Expression and purification of fusion proteins

Basic Protocol 2: Analysis of prey/bait pairs by SLC-based NS2B-NS3 interaction assay

Support Protocol 1: Interaction specificity assay

Support Protocol 2: Competition binding assay: Dose-response inhibition using cold prey or bait

Support Protocol 3: Competition binding assay: Inhibition by MBP-NS3 versus irrelevant MBP tag

Support Protocol 4: SLC-based NS2B-NS3 interaction assay using NS2B mutations known to disrupt NS2B-NS3 interactions

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分裂荧光素酶互补法分析蛋白-蛋白相互作用

蛋白质之间的相互作用在人类疾病中很重要。开发和完善工具来理解信号蛋白之间的物理接触是至关重要的。本文描述了一种分裂荧光素酶互补(SLC)方法，旨在发现蛋白质-蛋白质相互作用的抑制剂。构建、表达和纯化具有分裂荧光素酶的不同融合蛋白，然后评估以确定产生最强发光的最佳对。进一步证实了SLC的特异性和亲和力。以NS2B-NS3相互作用为例，提供了执行这些测定的分步说明。NS2B是黄病毒NS3蛋白酶功能的重要辅因子。进一步讨论了这些检测方法的优缺点。©2019 by John Wiley &基本方案1:融合蛋白的表达和纯化基本方案2:基于slc的NS2B-NS3相互作用分析猎物/诱饵对支持方案1:相互作用特异性分析支持方案2:竞争结合试验:使用冷猎物或诱饵进行剂量反应抑制支持方案3:竞争结合试验:MBP- ns3对不相关MBP标记的抑制作用支持方案4:基于slc的NS2B- ns3相互作用实验，使用已知的破坏NS2B- ns3相互作用的NS2B突变

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Current protocols in toxicology

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