tetano
Editor, Senior Moderator
Sci Rep
. 2022 Apr 29;12(1):7010.
doi: 10.1038/s41598-022-11053-7.
Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
Samuel S Slattery[SUP] 1 [/SUP], Daniel J Giguere[SUP] 1 [/SUP], Emily E Stuckless[SUP] 1 [/SUP], Arina Shrestha[SUP] 1 [/SUP], Lee-Ann K Briere[SUP] 1 [/SUP], Alexa Galbraith[SUP] 2 [/SUP], Stephen Reaume[SUP] 2 [/SUP], Xenia Boyko[SUP] 1 [/SUP], Henry H Say[SUP] 1 [/SUP], Tyler S Browne[SUP] 1 [/SUP], Mallory I Frederick[SUP] 1 [/SUP], Jeremy T Lant[SUP] 1 [/SUP], Ilka U Heinemann[SUP] 1 [/SUP], Patrick O'Donoghue[SUP] 1 3 [/SUP], Liann Dsouza[SUP] 4 [/SUP], Steven Martin[SUP] 4 [/SUP], Peter Howard[SUP] 4 [/SUP], Christopher Jedeszko[SUP] 5 [/SUP], Kinza Ali[SUP] 5 [/SUP], Garth Styba[SUP] 5 [/SUP], Martin Flatley[SUP] 6 [/SUP], Bogumil J Karas[SUP] 1 [/SUP], Gregory B Gloor[SUP] 7 [/SUP], David R Edgell[SUP] 8 [/SUP]
Affiliations
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.
. 2022 Apr 29;12(1):7010.
doi: 10.1038/s41598-022-11053-7.
Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics
Samuel S Slattery[SUP] 1 [/SUP], Daniel J Giguere[SUP] 1 [/SUP], Emily E Stuckless[SUP] 1 [/SUP], Arina Shrestha[SUP] 1 [/SUP], Lee-Ann K Briere[SUP] 1 [/SUP], Alexa Galbraith[SUP] 2 [/SUP], Stephen Reaume[SUP] 2 [/SUP], Xenia Boyko[SUP] 1 [/SUP], Henry H Say[SUP] 1 [/SUP], Tyler S Browne[SUP] 1 [/SUP], Mallory I Frederick[SUP] 1 [/SUP], Jeremy T Lant[SUP] 1 [/SUP], Ilka U Heinemann[SUP] 1 [/SUP], Patrick O'Donoghue[SUP] 1 3 [/SUP], Liann Dsouza[SUP] 4 [/SUP], Steven Martin[SUP] 4 [/SUP], Peter Howard[SUP] 4 [/SUP], Christopher Jedeszko[SUP] 5 [/SUP], Kinza Ali[SUP] 5 [/SUP], Garth Styba[SUP] 5 [/SUP], Martin Flatley[SUP] 6 [/SUP], Bogumil J Karas[SUP] 1 [/SUP], Gregory B Gloor[SUP] 7 [/SUP], David R Edgell[SUP] 8 [/SUP]
Affiliations
- PMID: 35487958
- DOI: 10.1038/s41598-022-11053-7
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.