Zhou Tongqing, Teng I-Ting, Olia Adam S, Cerutti Gabriele, Gorman Jason, Nazzari Alexandra, Shi Wei, Tsybovsky Yaroslav, Wang Lingshu, Wang Shuishu, Zhang Baoshan, Zhang Yi, Katsamba Phinikoula S, Petrova Yuliya, Banach Bailey B, Fahad Ahmed S, Liu Lihong, Lopez Acevedo Sheila N, Madan Bharat, de Souza Matheus Oliveira, Pan Xiaoli, Wang Pengfei, Wolfe Jacy R, Yin Michael, Ho David D, Phung Emily, DiPiazza Anthony, Chang Lauren, Abiona Olubukula, Corbett Kizzmekia S, DeKosky Brandon J, Graham Barney S, Mascola John R, Misasi John, Ruckwardt Tracy, Sullivan Nancy J, Shapiro Lawrence, Kwong Peter D
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
These authors contributed equally.
bioRxiv. 2020 Jun 23:2020.06.22.166033. doi: 10.1101/2020.06.22.166033.
Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed 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 included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized 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.
包含严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白特定区域的生物素标记分子探针，将有助于分离和鉴定靶向这种新出现病原体的抗体。为了开发此类探针，我们设计了构建体，其包含N端纯化标签、位点特异性蛋白酶切割位点、感兴趣的探针区域以及生物素连接酶靶向的C端序列。探针区域包括全长刺突胞外域以及各种亚区域，并且我们还设计了突变体以消除对血管紧张素转换酶2（ACE2）受体的识别。瞬时转染产生的生物素标记探针产量范围从完整胞外域的约0.5 mg/L到几个亚区域的>5 mg/L。对探针的抗原性和ACE2识别进行了表征，并且通过冷冻电子显微镜确定了刺突胞外域探针的结构。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，能够实现SARS-CoV-2刺突胞外域探针的简化开发。
