Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

• Published in: bioRxiv 2020-11
• Main Authors: Mehalko, Jennifer, Drew, Matthew, Snead, Kelly, Denson, John-Paul, Wall, Vanessa, Taylor, Troy, Sadtler, Kaitlyn, Messing, Simon, Gillette, William, Esposito, Dominic
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Antigens; Cell culture; Chromatography; Deoxyribonucleic acid; DNA; Enzyme-linked immunosorbent assay; Gel electrophoresis; Investigations; Metal ions; Molecular weight; Polyacrylamide; Protein purification; Proteins; Serology; Severe acute respiratory syndrome coronavirus 2; Signal peptides; Sodium lauryl sulfate; Spike protein; Streptavidin
• ISSN: 2692-8205
• DOI: 10.1101/2020.11.18.388868

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: Improved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parametersTwo versions of RBD show different sensitivity in serology assaysYields of greater than 50 mg/l obtained under optimal conditionsMagnetic bead purification technology improves throughput of protein production.