Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.
Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.
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DOI：
10.1021/acsomega.0c03512
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发表时间：
2021-01-12
期刊：
影响因子：
4.1
通讯作者：
Almo SC
中科院分区：
文献类型：
--
作者：
Herrera NG;Morano NC;Celikgil A;Georgiev GI;Malonis RJ;Lee JH;Tong K;Vergnolle O;Massimi AB;Yen LY;Noble AJ;Kopylov M;Bonanno JB;Garrett-Thomson SC;Hayes DB;Bortz RH 3rd;Wirchnianski AS;Florez C;Laudermilch E;Haslwanter D;Fels JM;Dieterle ME;Jangra RK;Barnhill J;Mengotto A;Kimmel D;Daily JP;Pirofski LA;Chandran K;Brenowitz M;Garforth SJ;Eng ET;Lai JR;Almo SC
冠状病毒病2019(新冠肺炎)是由新型严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)引起的全球性健康危机，迫切需要大量生产高质量的SARS-CoV-2 Spike(S)蛋白，用于临床和基础科学设置。为了满足这一需求，我们评估了先前报道的两个S蛋白结构在Expi293F和ExpiCHO-S细胞中的表达和纯化，这两个不同的细胞系被选为蛋白表达增加的两个不同细胞系。我们发现，在ExpiCHO-S细胞中，SARS-CoV-2 S蛋白的产量都有所提高。对两种细胞生产的SARS-CoV-2 S蛋白的生化、生物物理和结构(冷冻-EM)分析表明，所报道的纯化策略获得了高质量的S蛋白(非聚集、均匀的物质，具有适当的生化和生物物理性质)，对20种沉积的S蛋白的冷冻-EM结构分析表明，由614-642和828-854氨基酸组成的区域具有构象可塑性。重要的是，我们证明了这两种重组S蛋白的不同制备方法在两种不同的血清学检测中表现出相同的行为。我们还通过检测与人分泌组中拟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，没有报道新的结合伙伴，尤其是未能验证Spike：CD147的相互作用。此外，这些蛋白质的抗原性通过标准的ELISA和灵活的蛋白质微阵列形式得到证明。我们共同建立了一系列指标，以确保生产高质量的S蛋白，以支持临床、生物、生化、结构和机制研究，以抗击由SARS-CoV-2引起的全球大流行。
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.
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