Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada.
Curr Protoc. 2024 Mar;4(3):e1007. doi: 10.1002/cpz1.1007.
An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.
已开发出一种优化的方案，用于从严重急性呼吸系统综合征冠状病毒 2 型（SARS-CoV-2）中表达和纯化核心 RNA 依赖性 RNA 聚合酶（RdRP）复合物。由于 nsp12 的复杂多域折叠以及涉及 nsp7、nsp8 和 nsp12 的多聚体复合物的组装，因此活性核心 SARS-CoV-2 RdRp 复合物的表达和纯化具有挑战性。我们的方法采用了先前发表的方法，在大肠杆菌中表达核心 SARS-CoV-2 RdRP 复合物，并将其与表达 nsp12 与麦芽糖结合蛋白融合的程序相结合，以促进具有酶活性的核心聚合酶复合物的有效组装和纯化。该方法提供了一个可靠的平台，可生产毫克量的聚合酶复合物，其 nsp7、nsp8 和 nsp12 的预期比例分别为 1:2:1，并且去除了所有亲和标签。与之前报道的方法相比，该方法解决了一些限制，为使用已在广泛可访问的 Addgene 存储库中存放的重组质粒构建体进行 SARS-CoV-2 RdRP 复合物的结构、功能和抑制研究提供了可靠的活性聚合酶复合物来源。© 2024 作者。 Wiley 期刊出版公司出版的《当代协议》。基本方案 1：在大肠杆菌细胞中表达和生产 SARS-CoV-2 nsp7、nsp8 和 nsp12 支持方案：昆虫细胞培养物的建立和维持基本方案 2：在 Sf9 细胞中生成重组杆状病毒和在 T. ni 细胞中生产 nsp12 融合蛋白基本方案 3：SARS-CoV-2 核心聚合酶复合物的纯化。
