Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
| Autor: | Matthew Drew, Vanessa Wall, Kaitlyn Sadtler, Troy Taylor, Dominic Esposito, William K. Gillette, John-Paul Denson, Kelly Snead, Simon Messing, Jennifer Mehalko |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
Signal peptide TFF tangential flow filtration Population Enzyme-Linked Immunosorbent Assay Antibodies Viral AnSEC analytical size exclusion chromatography 01 natural sciences Article Serology 03 medical and health sciences chemistry.chemical_compound RBD receptor binding domain Antigen Protein Domains 010608 biotechnology Protein biosynthesis Protein production Humans IMAC immobilized metal ion affinity chromatography education CV column volume 030304 developmental biology 0303 health sciences education.field_of_study Chemistry SARS-CoV-2 SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis COVID-19 Promoter ELISA enzyme-linked immunosorbent assay MWCO molecular weight cut-off SBP streptavidin-binding peptide Receptor-binding domain Biochemistry Cell culture Spike Glycoprotein Coronavirus ELISA SEC size exclusion chromatography DNA Protein Binding Biotechnology |
| Zdroj: | Protein Expression and Purification bioRxiv article-version (status) pre article-version (number) 1 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2020.105802 |
| Popis: | 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 parameters. • Two versions of RBD show different sensitivity in serology assays. • Yields of greater than 50 mg/l obtained under optimal conditions. • Magnetic bead purification technology improves throughput of protein production. |
| Databáze: | OpenAIRE |
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