Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation

Tingting Li; Qingbing Zheng; Hai Yu; Dinghui Wu; Wenhui Xue; Yuyun Zhang; Xiaofen Huang; Lizhi Zhou; Zhigang Zhang; Zhenghui Zha; Tingting Chen; Zhiping Wang; Jie Chen; Hui Sun; Tingting Deng; Yingbin Wang; Yixin Chen; Qinjian Zhao; Jun Zhang; Ying Gu; Shaowei Li; Ningshao Xia

The recombinant proteins were mostly soluble expressed and secreted into the 113 culture medium. The centrifugation supernatants of cell culture went through metal 114 affinity chromatography using Ni-NTA resin. S, S1 and RBD proteins were mainly as detection antibody (Fig. 1C) . Interestingly, about one half S proteins were cleaved 119 into S1 (identical migration site to S1 lane in Fig. 1C ) and S2 (about 80kDa developed 120 in anti-His WB) possibly by innate furin of insect cell that was also found in other cases (Fig. 1D ). These peaks fractionated at retention volume 28mL, 36mL, 48mL, 124 and 65mL, were further harvested and subjected to SDS-PAGE analysis. The results 125 indicated that S proteins together with cleaved S1/S2 were resolved at peak 1 in size-126 exclusion chromatography (Fig. 1D ) and showed a high purity of over 95% total 127 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.16.994152 doi: bioRxiv preprint S/S1/S2 in gel (Fig. 1E ). Overall, one-step Ni-NTA affinity chromatography produced 128 RBD with 95% purity and a yield of 30 mg per L cell culture, S1 with about 90% purity 129 and 10 mg per L yield, while further purification through a size-exclusion 130 chromatography (SEC), the resultant S sample had over 95% purity regarding intact S 131 and cleaved S1/S2, and was harvested in a yield of 1 mg per L cell culture. These data The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.16.994152 doi: bioRxiv preprint molecular weight of intact S trimer. The three proteins were further analyzed by 150 differential scanning calorimetry (DSC) that was usually used to investigate the inner 151 thermostability of macromolecules or their complexes 14 . RBD and S1 showed one 152 major peak at comparable thermal denaturation midpoints (Tm) of 46.0 °C and 45.5 °C, 153 respectively ( Fig. 3G and 3H ), whereas S sample showed two major peaks at Tm of 154 45.5°C (identical to Tm of S1) and 64.5°C (Fig. 3I) , which might reflect the coexistence 155 of intact S and cleaved S1/S2.