The Nuts and Bolts of SARS-CoV-2 Spike Receptor-Binding Domain Heterologous Expression
Mariano Maffei, Linda Celeste Montemiglio, Grazia Vitagliano, Luigi Fedele, Shaila Sellathurai, Federica Bucci, Mirco Compagnone, Valerio Chiarini, Cécile Exertier, Alessia Muzi, Giuseppe Roscilli, Beatrice Vallone, Emanuele Marra
Abstract
COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.