Abstract
The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the eukaryotic microalgae Phaeodactylum tricornutum to develop an inducible overexpression system for SARS-CoV-2 proteins. Limiting phosphate and iron in growth media induced expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the P. tricornutum HASP1 promoter in the wild-type strain and in a histidine auxotrophic strain that alleviates the requirement for antibiotic selection of expression plasmids. The RBD was purified from whole cell extracts (algae-RBD) with yield compromised by the finding that 90-95% of expressed RBD lacked the genetically encoded C-terminal 6X-histidine tag. Constructs that lacked the TEV protease site between the RBD and C-terminal 6X-histidine tag retained the tag, increasing yield. Purified algae-RBD was found to be N-linked glycosylated by treatment with endoglycosidases, was cross-reactive with anti-RBD polyclonal antibodies, and inhibited binding of recombinant RBD purified from mammalian cell lines to the human ACE2 receptor. We also show that the algae-RBD can be used in a lateral flow assay device to detect SARS-CoV-2 specific IgG antibodies from donor serum at sensitivity equivalent to assays performed with RBD made in mammalian cell lines. Our study shows that P. tricornutum is a scalable system with minimal biocontainment requirements for the inducible production of SARS-CoV-2 or other coronavirus antigens for pandemic diagnostics.
【초록키워드】 Treatment, SARS-CoV-2, coronavirus, pandemic, Therapeutics, COVID-19 pandemic, iron, diagnostics, Betacoronavirus, Spike protein, Antigen, lateral flow, serum, sensitivity, Receptor-binding domain, RBD, SARS-CoV-2 spike protein, Synthetic biology, polyclonal antibodies, expression, Donor, binding, antibiotic, SARS-CoV-2 proteins, Plasmid, Biomedical research, anti-RBD, cross-reactive, histidine, phosphate, type strain, Viral antigen, specific IgG antibodies, expression plasmids, wild-type strain, human ACE2 receptor, finding, overexpression, promoter, growth media, yield, mammalian cell, approach, Cell, performed, detect, develop, caused, inhibited, can be used, expressed, the RBD, the receptor-binding domain, retained, purified, C-terminal, glycosylated, eukaryotic, alleviate, specific IgG antibody, TEV protease, the SARS-CoV-2, 【제목키워드】 pandemic, diagnostics, expression, domain, the SARS-CoV-2,