The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L⁻¹ culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

中文翻译：

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SARS-CoV-2 刺突蛋白受体结合域在哺乳动物细胞中的表达和纯化，用于免疫分析

SARS-CoV-2 病毒刺突糖蛋白的受体结合域 (RBD) 介导与宿主细胞的相互作用，是病毒内化所必需的。因此，它是中和抗体的主要目标。受体结合域很快成为 COVID-19 研究以及诊断工具和新一代疫苗开发的主要目标。在这里，我们提供了从瞬时转染的 Expi293F 细胞中高产表达和一步亲和纯化重组 RBD 的详细方案。Expi293F 哺乳动物细胞可以在特殊配制的悬浮培养无血清培养基中生长至极高密度，这使其成为分泌蛋白生产的绝佳工具。高度纯化的 RBD 被糖基化，结构完整，并形成同聚复合物。通过这种快速简便的方法，我们能够生产大量 RBD（80 mg·L ^-1培养物），我们已成功地将其用于免疫测定，以检查基于 mRNA 的小鼠疫苗接种后的抗体滴度和血清转化。