Herrera Natalia G, Morano Nicholas C, Celikgil Alev, Georgiev George I, Malonis Ryan J, Lee James H, Tong Karen, Vergnolle Olivia, Massimi Aldo B, Yen Laura Y, Noble Alex J, Kopylov Mykhailo, Bonanno Jeffrey B, Garrett-Thomson Sarah C, Hayes David B, Bortz Robert H, Wirchnianski Ariel S, Florez Catalina, Laudermilch Ethan, Haslwanter Denise, Fels J Maximilian, Dieterle M Eugenia, Jangra Rohit K, Barnhill Jason, Mengotto Amanda, Kimmel Duncan, Daily Johanna P, Pirofski Liise-Anne, Chandran Kartik, Brenowitz Michael, Garforth Scott J, Eng Edward T, Lai Jonathan R, Almo Steven C
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461, United States.
National Resource for Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 89 Convent Ave., New York, New York 10027, United States.
ACS Omega. 2020 Dec 21;6(1):85-102. doi: 10.1021/acsomega.0c03512. eCollection 2021 Jan 12.
Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.
2019冠状病毒病（COVID-19）是由新型严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引起的全球健康危机，迫切需要大量生产高质量的SARS-CoV-2刺突（S）蛋白，用于临床和基础科学研究。为满足这一需求，我们评估了两种先前报道的S蛋白构建体在Expi293F和ExpiCHO-S细胞（两种为提高蛋白表达而选择的不同细胞系）中的表达和纯化情况。我们发现ExpiCHO-S细胞能提高两种SARS-CoV-2 S蛋白的产量。对这两种细胞系产生的SARS-CoV-2 S蛋白进行的生化、生物物理和结构（冷冻电镜）表征表明，所报道的纯化策略能产生高质量的S蛋白（非聚集、具有适当生化和生物物理特性的均匀物质），对20个已存入的S蛋白冷冻电镜结构的分析揭示了由氨基酸614 - 642和828 - 854组成的区域存在构象可塑性。重要的是，我们表明来自任一细胞系的这两种重组S蛋白的多个制备物在两种不同的血清学检测中表现出相同的行为。我们还通过检查与人类分泌组中提议的结合伙伴的相互作用来评估S蛋白介导的宿主细胞结合的特异性，未发现新的结合伙伴，尤其未能验证刺突蛋白与CD147的相互作用。此外，这些蛋白的抗原性通过标准酶联免疫吸附测定（ELISA）和灵活的蛋白质微阵列形式得以证明。总体而言，我们建立了一系列指标，以确保生产高质量的S蛋白，支持针对由SARS-CoV-2引起的全球大流行的临床、生物学、生化、结构和机制研究。
