| An in vitro Transcription/translation System for Detection of Protein Interaction
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Author:
Date:
2016-05-05
[Abstract] Studying protein-protein interaction is crucial to understand the fundamental processes of molecular biology. High-throughput screening, such as immunoprecipitation followed by proteomic analysis, allows for the identification of numerous candidate partners that might interact with a selected protein. However, experimental validation of protein-protein interaction requires conventional cloning and recombinant protein expression/purification, which are complicated and labor-intensive techniques. Here, we demonstrate an efficient experimental pipeline for verifying protein-protein interactions between a bait protein using the example of Odontoglossum ringspot virus (ORSV) capsid protein (CP) and the host CP-binding protein. These candidate CP-binding proteins were identified ...
[摘要] 研究蛋白质 - 蛋白质相互作用对于理解分子生物学的基本过程是至关重要的。高通量筛选，如免疫沉淀，然后蛋白质组分析，允许鉴定可能与选择的蛋白质相互作用的许多候选伙伴。然而，蛋白质 - 蛋白质相互作用的实验验证需要常规克隆和重组蛋白表达/纯化，这是复杂和劳动密集型技术。在这里，我们演示了一个有效的实验管道，用于验证使用Odontoglossum环斑病毒（ORSV）衣壳蛋白（CP）和宿主CP结合蛋白的例子的诱饵蛋白之间的蛋白质 - 蛋白质相互作用。这些候选CP结合蛋白通过高通量蛋白质组学和转录组学方法进行鉴定。使用TOPO克隆策略，将每个候选基因克隆到表达载体中，用于在体外转录/翻译系统的单个步骤中表达His标记的重组蛋白。这种表达的His标记的候选物可以在共免疫沉淀（co-IP）测定中用作CP诱饵蛋白的猎物，以验证它们的物理相互作用。不需要传统的蛋白质表达和纯化，该管道简化了验证过程，并为高通量蛋白质 - 蛋白质相互作用研究提供了解决方案。
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| Purification and Detection of a PDGA Depolymerase from Pusillimonas noertemannii
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Author:
Date:
2014-11-05
[Abstract] The purification of a target protein from a complex mixture of proteins is a challenging undertaking. If the target protein has been previously characterised, then information such as subcellular location, function, molecular weight and pI can be used for the design of a purification strategy. However, if the target protein is uncharacterised or little information regarding its characteristics is available, a generic purification protocol can be employed that is optimised as additional characteristics of the target protein are determined during subsequent purification steps. Herein, we describe the protocol for the purification and detection of a poly-γ-D-glutamic acid (PDGA) depolymerase from a consortium culture of two Gram-negative bacteria using a combination of chromatography, ...
[摘要] 从蛋白质的复杂混合物中纯化靶蛋白是一项具有挑战性的任务。 如果靶蛋白以前已被表征，则诸如亚细胞定位，功能，分子量和pI的信息可用于设计纯化策略。 然而，如果目标蛋白质是未表征的或者关于其特征的很少的信息可用，则可以使用通用的纯化方案，其随着在随后的纯化步骤期间测定靶标蛋白质的附加特征而被优化。 在这里，我们描述了从两个革兰氏阴性细菌的联合培养物使用色谱，2D电泳和酶谱的组合纯化和检测聚-γ-D-谷氨酸（PDGA）解聚酶的协议。
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