Unit of Structural Dynamics of Macromolecules, Institut Pasteur & CNRS UMR, Paris, France.
Department of Molecular Biology and Genetics, Faculty of Science, Van Yüzüncü Yıl University, Van, Turkey.
PLoS One. 2021 Apr 29;16(4):e0250610. doi: 10.1371/journal.pone.0250610. eCollection 2021.
To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome's transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451).
为了阻止由严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）引起的 COVID-19 大流行，迄今为止，已经导致超过 250 万人死亡，因此急需新的抗病毒分子。SARS-CoV-2 的复制需要 RNA 依赖性 RNA 聚合酶（RdRp），这使得 RdRp 成为抗病毒药物的理想靶点。RdRp 是由 3 种病毒蛋白（nsp7、nsp8 和 nsp12）组成的多亚基复合物，可确保~30kb RNA 基因组的转录和复制。迄今为止，用于 RdRp 过量生产的主要策略包括分别表达和纯化这三个亚基，然后在体外组装复合物。然而，nsp12 在细菌表达系统中的溶解度有限，通常在昆虫细胞中产生。在这里，我们描述了一种在大肠杆菌中共同表达全长 SARS-CoV-2 RdRp 的替代策略，使用单个质粒。对纯化的重组 SARS-CoV-2 RdRp 的特性进行了表征，表明它形成了预期的（nsp7）（nsp8）2（nsp12）化学计量比的复合物。使用引物延伸测定法测量了 RNA 聚合酶活性，表明纯化的酶具有功能。该纯化方案可以在一天内完成，比所有其他已发表的方案都快。我们的构建体非常适合针对非常大的化合物文库筛选 RdRp 及其变体，并且已经通过 Addgene 质粒库（Addgene ID：165451）提供给科学界。
