Expression, purification, and refolding of an optimized SARS-CoV-2 receptor binding domain in E. coli.

The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a significant risk to global health, warranting the formulation of efficient preventive and therapeutic measures to tackle its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of coronaviruses plays a pivotal role in viral attachment and entry into host cells. The receptor-binding domain (RBD) of the SARS-CoV-2 S protein has demonstrated a robust binding affinity to ACE2 receptors in humans. Consequently, it has become a prime target for therapeutic interventions using antibodies, vaccines, or other designed inhibitors. We engineered an RBD sequence with refined ORF boundaries guided by structural insights, which enabled efficient in vitro refolding. This highlights the critical role of precise expression cassette design in a plasmid, extending beyond conventional parameters such as promoter or fusion tag selection. Using customized refolding procedures, we obtained 10-12 mg of active protein from a one-liter LB culture. The biological activity of the refolded RBD was confirmed by monitoring its interaction with the designed LCB1 miniprotein ligand by surface plasmon resonance, wherein they exhibited significant affinity levels as reflected by their dissociation constants (KD values <10 nM). The resulting RBD could be an ideal target for designing potent COVID-19 antivirals.