Sheffield Institute for Nucleic Acids and Florey Institute, Section of Infection and Immunity, Division of Clinical Medicine, School of Medicine and Population Health, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, U.K.
Michael Barber Centre for Collaborative Mass Spectrometry, Department of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Biochem J. 2024 Jun 5;481(11):669-682. doi: 10.1042/BCJ20240019.
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.
自 2019 年末 COVID-19 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）的基本生物学特性、其在诊断检测中的应用及其作为疫苗成分的潜在应用受到了广泛关注。在这里，我们报告了在大肠杆菌中可规模化表达和纯化可溶性、免疫活性 SARS-CoV-2 Ncap。我们将编码原始 Ncap 序列和带有 N 端 6His 亲和标签（序列 MHHHHHHG）的四个常见变异体的优化密码子合成基因克隆到带有受 lac 操纵子结合位点调控的噬菌体 T5 合成启动子的诱导表达载体中。这些构建体用于表达 Ncap 蛋白，并开发了有效的纯化方案，可从每升培养物中获得超过 200mg 的纯化 Ncap。这些蛋白被用于 ELISA 检测，以比较它们对人血清的反应。我们的结果表明，带有 6His 标签和未标记的原始 Ncap 蛋白之间没有检测到差异，但其他 Ncap 分离株的血清敏感性可能略有降低。
