Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.
Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.
复制标题
DOI：
10.1016/j.celrep.2020.108322
复制
发表时间：
2020-10-27
期刊：
影响因子：
8.8
通讯作者：
Kwong PD
中科院分区：
文献类型：
--
作者：
Zhou T;Teng IT;Olia AS;Cerutti G;Gorman J;Nazzari A;Shi W;Tsybovsky Y;Wang L;Wang S;Zhang B;Zhang Y;Katsamba PS;Petrova Y;Banach BB;Fahad AS;Liu L;Lopez Acevedo SN;Madan B;Oliveira de Souza M;Pan X;Wang P;Wolfe JR;Yin M;Ho DD;Phung E;DiPiazza A;Chang LA;Abiona OM;Corbett KS;DeKosky BJ;Graham BS;Mascola JR;Misasi J;Ruckwardt T;Sullivan NJ;Shapiro L;Kwong PD
生物素标记的分子探针包含严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)尖峰的特定区域，将有助于分离和鉴定针对这种新出现的病原体的抗体。在这里，我们设计了包含一个N端纯化标签、一个特定部位的蛋白酶裂解位点、感兴趣的探针区和一个以生物素连接酶为靶点的C端序列的构建体。探针区包括全长的尖峰结构域以及不同的亚区，我们还设计了突变体来消除对血管紧张素转换酶2(ACE2)受体的识别。瞬时转染的生物素标记探针的产量范围从完整胞外结构域的∼0.5m g/L到几个亚区的>；5 mg/L。对探针的抗原性和ACE2识别进行了表征，并通过冷冻电子显微镜确定了钉状胞外结构域探针的结构。我们还表征了生物素化探针的抗体结合特异性和细胞分选能力。总之，基于结构的设计与高效的纯化和生物素化过程相结合，可以简化SARS-CoV-2尖状胞外结构域探针的开发。基于结构的SARS-CoV-2棘突外区和亚区探针的柱上生物素化和纯化是通过亲和标签和HRV3C裂解策略实现的。通过“剪切和粘贴”策略发展多样化的分子探针探针在SARS-CoV-2抗体发现和免疫评估中的应用SARS-CoV-2棘突探针是抗体发现和疫苗评价的关键。通过使用基于结构的设计、亲和纯化、柱上生物素化和序列特异性蛋白酶切割，周等人。设计一种策略，允许快速开发用于B细胞分选、抗体发现和免疫评估的SARS-CoV-2尖峰探针。
Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes. Structure-based design of SARS-CoV-2 spike ectodomain and subdomain probes On-column biotinylation and purification is enabled by affinity tag and HRV3C cleavage Development of diverse molecular probes by “cut-and-paste” strategy Application of probes to SARS-CoV-2 antibody discovery and immune evaluation SARS-CoV-2 spike probes are key to antibody discovery and vaccine evaluation. By using structure-based design, affinity purification, on-column biotinylation, and sequence-specific protease cleavage, Zhou et al. devise a strategy that allows for the rapid development of SARS-CoV-2 spike probes for B cell sorting, antibody discovery, and immune assessment.
登录
查看更多内容
影响因子：
48
作者：
Barad BA;Echols N;Wang RY;Cheng Y;DiMaio F;Adams PD;Fraser JS
通讯作者：
Fraser JS
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
168.9
作者：
Gaudinski, Martin R.;Coates, Emily E.;Ledgerwood, Julie E.
通讯作者：
Ledgerwood, Julie E.
影响因子：
82.9
作者：
Zost SJ;Gilchuk P;Chen RE;Case JB;Reidy JX;Trivette A;Nargi RS;Sutton RE;Suryadevara N;Chen EC;Binshtein E;Shrihari S;Ostrowski M;Chu HY;Didier JE;MacRenaris KW;Jones T;Day S;Myers L;Eun-Hyung Lee F;Nguyen DC;Sanz I;Martinez DR;Rothlauf PW;Bloyet LM;Whelan SPJ;Baric RS;Thackray LB;Diamond MS;Carnahan RH;Crowe JE Jr
通讯作者：
Crowe JE Jr
DOI：
10.1038/nrmicro3143
发表时间：
2013-12
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
通讯作者：
--