Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System

Abstract

The spike protein (SP) of SARS-CoV-2 is an important target for COVID-19 therapeutics and vaccines as it binds to the ACE2 receptor and enables viral infection. Rapid production and functional characterization of properly folded SP is of the utmost importance for studying the immunogenicity and receptor-binding activity of this protein considering the emergence of highly infectious viral variants. In this study, we attempted to express the receptor-binding region (RBD) of SARS-CoV-2 SP containing disulfide bonds using the wheat germ cell-free protein synthesis system. By adding protein disulfide isomerase (PDI) and endoplasmic reticulum oxidase (ERO1α) to the translational reaction mixture, we succeeded in synthesizing a functionally intact RBD protein that can interact with ACE2. Using this RBD protein, we have developed a high-throughput AlphaScreen assay to evaluate the RBD–ACE2 interaction, which can be applied for drug screening and mutation analysis. Thus, our method sheds new light on the structural and functional properties of SARS-CoV-2 SP and has the potential to contribute to the development of new COVID-19 therapeutics.

Quantitative assessment of multiple pathogen exposure and immune dynamics at scale

“…We first tested a three-antigen MISPA assay that targeted two SARS-CoV-2 antigens of the original Wuhan strain, receptor-binding domain (wRBD) of the spike protein and nucleocapsid (NC), along with a negative control protein, the green fluorescent protein (GFP). The wRBD is a glycosylated protein with four disulfide bonds, and serum samples from COVID-19 patients showed only a weak response to IVTT-produced wRBD ( 13 ). Therefore, we expressed it in Expi293F cells.…”

“…Cell-based production is more cumbersome but necessary for some proteins. The majority of proteins were produced in MISPA using an IVTT coupled expression system, which has been employed extensively in the production of tens of thousands of proteins for use in serological studies ( 13 – 19 ). This HeLa cell lysate-based IVTT, which includes both human ribosomes and chaperone proteins, can produce folded and enzymatically active proteins ( 20 ).…”

SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing

“…Consistent with this, the RBD is a determinant of reduced S1/S2 proteolysis in the Omicron BA.1 spike [ 26 ], and the N-terminal domain of the Delta spike allosterically modulates S1/S2 processing [ 27 ]. Furthermore, similar to the C488 mutations in RBD, which disrupt the C480–C488 disulfide bond of the spike protein, Yamaoka et al recently showed that disulfide-bond formation is critical for RBD functioning, and C488A and C379A mutations lost their ACE2-binding ability [ 28 ]. In addition, Zhang et al showed that the C301F and C379F mutations eliminate the C291–C301 and C379–C432 disulfide bonds located in the S1 N-terminal domain and RBD, respectively, thus causing the S1/S2 processing loss of the spike protein [ 14 ].…”

SARS-Cov-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization And Intracellular S1/S2 Processing

“…Consistent with this, the RBD is a determinant of reduced S1/S2 proteolysis in the omicron BA.1 spike [21], and the N-terminal domain of the delta spike allosterically modulates S1/S2 processing [22]. Furthermore, similar to the C488 mutations in RBD, which disrupts the C480-C488 disulfide bond of the spike protein, Yamaoka et al recently showed that disulfide bond formation is critical for RBD functioning, and C488A and C379A mutations lost their ACE2-binding ability [23]. In addition, Zhang et al showed that the C301F and C379F mutations eliminate the C291-C301 and C379-C432 disulfide bonds located in the S1 N-terminal domain and RBD, respectively, thus causing S1/S2 processing loss of the spike protein [14].…”