The nuts and bolts of SARS-CoV-2 Spike Receptor Binding Domain heterologous expression

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 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.

Correlation of patient serum IgG, IgA and IgM antigen binding with COVID-19 disease severity using multiplexed SARS-CoV-2 antigen microarray and maintained relative IgA and IgM antigen binding over time

“…coli-expressed S1 [28]. E. coli-expressed non-glycosylated RBD protein was previous ly demonstrated to elicit approximately 7-fold lower binding by immunised rat serum IgG at 5 µg/mL in an ELISA compared to glycosylated RBD expressed in insect (Sf21) and HEK cells [40]. At lower concentrations, binding to E. coli-expressed RBD was below the assay threshold.…”

“…Proteins recombinantly expressed in E. coli are missing post-translational modificatio ns (PTMs) while those expressed in mammalian and insect cells can have PTMs, includ ing glycosylation, but the PTMs and glycosylation will vary depending on the system used with consequent impacts on protein functionality [40]. In human infections, the SARS-CoV-2 S protein is very heavily glycosylated with both high mannose and complex type N -linked glycosylation and O-linked glycosylation, which shields much of the viral protein backbone from host immune recognition including the enfolded RBD [41,42].…”

Biophysical fitness landscape of the SARS-CoV-2 Delta variant receptor binding domain

“…Spectra of the WT match those reported in the literature. 10,[37][38][39] More importantly, none of the mutations caused significant changes in the spectra, implying that the Delta mutations do not affect the global protein structure of RBD.…”

Addition of arginine hydrochloride and proline to the culture medium enhances recombinant protein expression in Brevibacillus choshinensis: The case of RBD of SARS-CoV-2 spike protein and its antibody

“…Since the beginning of the 2019 SARS-CoV-2 pandemic, RBD that is the key domain for human cell infection [ 19 , 20 ] has attracted much attention as a target molecule for treatment utilizing neutralizing antibodies [ 21 , 22 ] and vaccines [ 23 ]. In this context, recombinant expression of RBD in E. coli has been attempted; however, expression without the presence of any solubility-enhancing tag within the soluble fraction and with correct folding has not yet been achieved [ [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] ]. RBD possesses four disulfide bonds [ 33 ] that make it difficult for this domain to fold correctly.…”

“…RBD possesses four disulfide bonds [ 33 ] that make it difficult for this domain to fold correctly. Although many attempts have been made to refold RBDs from inclusion bodies, the procedure is complicated, and it has been suggested that the physicochemical properties may differ from those of proteins produced by baculovirus or by mammalian cell expression systems [ 31 ]. Although the RBD-MBP fusion protein was reported to be expressed in the soluble fraction of the cytoplasm [ 32 ], there was no structural or physicochemical information for the recombinant fusion protein except for information regarding ACE2 binding ability, and it may be possible that untagged RBD is insoluble when MBP is cleaved due to the strong solubilizing effect of MBP [ 34 ].…”