Wongnak Rawiwan, Brindha Subbaian, Yoshizue Takahiro, Onchaiya Sawaros, Mizutani Kenji, Kuroda Yutaka
Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8538, Japan.
Biophys Physicobiol. 2023 Sep 21;20(4):e200036. doi: 10.2142/biophysico.bppb-v20.0036. eCollection 2023.
Low-cost bacterial production of the receptor binding domain (RBD) of the SARS-CoV-2 Omicron spike protein holds significant potential in expediting the development of therapeutics against COVID-19. However, RBD contains eight cysteines forming four disulfide bonds, and expression in using standard protocols produces insoluble RBD forming non-native disulfide bonds. Here, we expressed RBD in T7 SHuffle with high aeration, which enhanced disulfide formation in the cytoplasm and reshuffling of non-native disulfide bonds, and at a low temperature of 16°C, which stabilized the native conformation and thus the formation of the native disulfide bonds. The yield of RBD was as high as 3 mg per 200 mL culture. We analyzed the conformational and biophysical properties of our -expressed RBD. First, the RP-HPLC elution profile indicated a single peak, suggesting that RBD was folded with a single disulfide bond pairing pattern. Next, circular dichroism analysis indicated a secondary structure content very close to that computed from the crystal structure. RBD's thermal denaturation monitored by CD was cooperative, strongly indicating a well-folded protein structure. Moreover, limited proteolysis showed that RBD was nearly as stable as RNase A, and the formation of native disulfide bonds was confirmed by LC-MS analysis. Furthermore, BLI analysis indicated a strong binding of RBD with the hACE2 with a dissociation constant of 0.83 nM, confirming the folded nature of RBD. Altogether, these results demonstrate that our -expression system can provide a large amount of highly purified RBD with correct disulfide bonds and native-like biochemical and biophysical properties.
低成本细菌生产严重急性呼吸综合征冠状病毒2（SARS-CoV-2）奥密克戎刺突蛋白的受体结合域（RBD）在加速抗2019冠状病毒病（COVID-19）治疗药物的开发方面具有巨大潜力。然而，RBD包含八个形成四个二硫键的半胱氨酸，使用标准方案在大肠杆菌中表达会产生形成非天然二硫键的不溶性RBD。在此，我们在大肠杆菌T7 SHuffle中高通气条件下表达RBD，这增强了细胞质中二硫键的形成以及非天然二硫键的重排，并且在16°C的低温下表达，这稳定了天然构象从而促进天然二硫键的形成。RBD的产量高达每200 mL培养物3 mg。我们分析了大肠杆菌表达的RBD的构象和生物物理特性。首先，反相高效液相色谱（RP-HPLC）洗脱图谱显示为单峰，表明RBD以单一二硫键配对模式折叠。其次，圆二色性分析表明二级结构含量与根据晶体结构计算得出的非常接近。通过圆二色性监测的RBD的热变性是协同的，强烈表明蛋白质结构折叠良好。此外，有限蛋白酶解表明RBD几乎与核糖核酸酶A一样稳定，并且通过液相色谱-质谱（LC-MS）分析证实了天然二硫键的形成。此外，生物层干涉（BLI）分析表明RBD与人类血管紧张素转换酶2（hACE2）有很强的结合，解离常数为0.83 nM，证实了RBD的折叠性质。总之，这些结果表明我们的大肠杆菌表达系统可以提供大量具有正确二硫键以及类似天然生化和生物物理特性的高度纯化的RBD。
