The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

中文翻译：

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严重急性呼吸综合征冠状病毒2核衣壳蛋白在大肠杆菌中的高效过表达和纯化


自 2019 年底 Covid19 大流行爆发以来，严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 核衣壳蛋白 (Ncap) 的基本生物学、其在诊断分析中的用途及其作为疫苗成分的潜在应用受到了广泛关注在此，我们报告了可溶性、免疫活性的 SARS-CoV-2 Ncap 在大肠杆菌中的可扩展表达和纯化。将编码原始 Ncap 序列和带有 N 端 6His 亲和标签（序列 MHHHHHHG）的四种常见变体的密码子优化合成基因克隆到携带由 lac 操纵子结合位点控制的调节噬菌体 T5 合成启动子的诱导型表达载体中。该构建体用于表达 Ncap 蛋白，所开发的方案可有效生产纯化的 Ncap，每升培养基的产量超过 200 毫克。这些蛋白质被用于 ELISA 测定，以比较它们对人类血清的反应。我们的结果表明，6His 标记和未标记的原始 Ncap 蛋白之间没有可检测到的差异，但血清对其他 Ncap 分离株的敏感性可能略有下降。