Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect approximately 50 million people, with over 1 million reported deaths, and a safe and effective vaccine remains urgently needed. Based on previous experience developing vaccines against SARS and MERS, we constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Collectively, these three recombinant protein RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity for their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, and had a lower tendency to form oligomers when compared to the other two proteins and was therefore selected for further vaccine development and evaluation.