Recombinant Expression and Improved Refolding of the SARS-CoV-2 Spike Receptor-Binding Domain in Escherichia coli.
TL;DR
An Escherichia coli platform to express the wild-type RBD was used as an initial model for the standardization of the methodology, demonstrating its adaptability to different RBD variants and supporting their use in serological assays as a rapid and scalable source of antigens.
Abstract
The receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein is central to serological diagnostics and vaccine development, but production in eukaryotic expression systems is costly and technically demanding. Here, we describe an Escherichia coli platform to express the wild-type RBD, used as an initial model for the standardization of the methodology. Subsequently, the platform was applied to the alpha and beta variants, demonstrating its adaptability to different RBD variants. Inclusion bodies were purified under denaturing conditions and refolded by size-exclusion chromatography using a linear urea gradient and a redox/anti-aggregation buffer that promotes correct disulfide-bond formation and minimizes aggregation. The procedure yielded ∼36 mg of soluble protein per liter of culture. The refolded recombinant RBDs of wild-type, alpha and beta variants, as well as RDB wild-type produced in Expi293 cells as a control were evaluated in ELISA assays against sera from healthy, vaccinated and convalescent SARS-CoV-2 patients. All recombinant RBDs produced in E. coli were recognized by patients' sera, demonstrating that they retain antigenicity and supporting their use in serological assays as a rapid and scalable source of antigens.