Abstract
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 E. coli using standard protocols produces insoluble RBD forming non-native disulfide bonds. Here, we expressed RBD in E. coli 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 E. coli-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 E. coli-expression system can provide a large amount of highly purified RBD with correct disulfide bonds and native-like biochemical and biophysical properties.