Bhattarai Matrika, Javaid Tasleem, Venkataraghavan Akshayaa, Faik Ahmed
Department of Environmental and Plant Biology, Ohio University, Athens, OH, USA.
Molecular and Cellular Biology program, Ohio University, Athens, OH, USA.
Bio Protoc. 2024 Sep 20;14(18):e5066. doi: 10.21769/BioProtoc.5066.
Progress in bioinformatics has facilitated the identification of a large number of putative glycosyltransferases (GTs) associated with many physiological processes. However, many of these GTs remain with unknown biochemical function due to numerous technical limitations. One of these limitations is the lack of innovative tools for large-scale screening of enzyme activity in vitro and testing protein-protein interactions (PPIs) between GT partners. Currently, testing the enzyme activity of a protein requires its production in a heterologous expression system and purification before enzyme assays, a process that is time-consuming and not amenable to high-throughput screening. To overcome this, we developed a platform called in vitro GT-array (-GT-ray). In this platform, 96-well microplates are coated with plasmid DNA encoding for tagged GTs and a capture antibody. Tagged GTs are produced from plasmid DNA via a cell-free in vitro transcription/translation (IVTT) system and captured through the anti-tag capture antibody directly on microplates. After washing to remove IVTT components, the captured enzymes can be considered purified, and their activity can be tested directly on microplates. The whole process can be performed in less than two days, compared to several weeks for currently available screening methods. The -GT-ray platform has also been adapted to investigate PPIs between GTs. Here, we provide a practical user guide for the preparation of GT-arrays coated with plasmid DNA and a capture antibody that can be used for monitoring enzyme activity and PPIs of GTs in a high-throughput manner. Key features • Synthesis of tagged proteins directly from plasmid DNA, which are captured by anti-tag antibody attached to microplates. • Captured tagged proteins can be considered as purified proteins ready for enzyme assays. • Our platform can be used for high-throughput screening of enzyme activity and protein-protein interactions in vitro in a short time. • Our platform can be used for biochemical characterization of difficult proteins such as membrane-integrated glycosyltransferases. • Our platform can be adapted to downstream analytical methods such as mass spectrometry (i.e., DPS-MS).
生物信息学的进展推动了大量与多种生理过程相关的假定糖基转移酶（GTs）的鉴定。然而，由于众多技术限制，这些GTs中的许多仍具有未知的生化功能。其中一个限制是缺乏用于体外大规模筛选酶活性以及测试GT伴侣之间蛋白质-蛋白质相互作用（PPI）的创新工具。目前，测试一种蛋白质的酶活性需要在异源表达系统中生产并在酶分析之前进行纯化，这一过程既耗时又不适用于高通量筛选。为了克服这一问题，我们开发了一个名为体外GT阵列（-GT-ray）的平台。在这个平台中，96孔微孔板用编码带标签GTs的质粒DNA和捕获抗体包被。带标签的GTs通过无细胞体外转录/翻译（IVTT）系统从质粒DNA产生，并通过抗标签捕获抗体直接在微孔板上捕获。洗涤以去除IVTT成分后，捕获的酶可被视为已纯化，其活性可直接在微孔板上进行测试。与目前可用的筛选方法需要数周时间相比，整个过程可在不到两天内完成。-GT-ray平台也已被用于研究GTs之间的PPI。在此，我们提供一份实用的用户指南，用于制备涂有质粒DNA和捕获抗体的GT阵列，该阵列可用于以高通量方式监测GTs的酶活性和PPI。关键特性 • 直接从质粒DNA合成带标签的蛋白质，这些蛋白质被附着在微孔板上的抗标签抗体捕获。 • 捕获的带标签蛋白质可被视为已纯化的蛋白质，可用于酶分析。 • 我们的平台可用于在短时间内对酶活性和蛋白质-蛋白质相互作用进行体外高通量筛选。 • 我们的平台可用于对诸如膜整合糖基转移酶等难处理蛋白质进行生化表征。 • 我们的平台可适用于下游分析方法，如质谱分析（即DPS-MS）。
