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Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics.
Slattery, Samuel S; Giguere, Daniel J; Stuckless, Emily E; Shrestha, Arina; Briere, Lee-Ann K; Galbraith, Alexa; Reaume, Stephen; Boyko, Xenia; Say, Henry H; Browne, Tyler S; Frederick, Mallory I; Lant, Jeremy T; Heinemann, Ilka U; O'Donoghue, Patrick; Dsouza, Liann; Martin, Steven; Howard, Peter; Jedeszko, Christopher; Ali, Kinza; Styba, Garth; Flatley, Martin; Karas, Bogumil J; Gloor, Gregory B; Edgell, David R.
  • Slattery SS; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Giguere DJ; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Stuckless EE; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Shrestha A; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Briere LK; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Galbraith A; Lambton College, 1457 London Rd, Sarnia, ON, N7S 6K4, Canada.
  • Reaume S; Lambton College, 1457 London Rd, Sarnia, ON, N7S 6K4, Canada.
  • Boyko X; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Say HH; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Browne TS; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Frederick MI; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Lant JT; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Heinemann IU; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • O'Donoghue P; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Dsouza L; Department of Chemistry, Western University, London, ON, N6A 3K7, Canada.
  • Martin S; Pond Technologies Inc., Markham, ON, L3R 9W7, Canada.
  • Howard P; Pond Technologies Inc., Markham, ON, L3R 9W7, Canada.
  • Jedeszko C; Pond Technologies Inc., Markham, ON, L3R 9W7, Canada.
  • Ali K; International Point of Care Inc., 135 The West Mall Unit 9, Toronto, ON, M9C 1C2, Canada.
  • Styba G; International Point of Care Inc., 135 The West Mall Unit 9, Toronto, ON, M9C 1C2, Canada.
  • Flatley M; International Point of Care Inc., 135 The West Mall Unit 9, Toronto, ON, M9C 1C2, Canada.
  • Karas BJ; Suncor Energy Inc., Sarnia Refinery, 1900 River Road, Sarnia, ON, N7T 7J3, Canada.
  • Gloor GB; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
  • Edgell DR; Department of Biochemistry, Schlich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada. ggloor@uwo.ca.
Sci Rep ; 12(1): 7010, 2022 04 29.
Article in English | MEDLINE | ID: covidwho-1890243
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Diatoms / COVID-19 Type of study: Diagnostic study / Randomized controlled trials Limits: Animals / Humans Language: English Journal: Sci Rep Year: 2022 Document Type: Article Affiliation country: S41598-022-11053-7

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Diatoms / COVID-19 Type of study: Diagnostic study / Randomized controlled trials Limits: Animals / Humans Language: English Journal: Sci Rep Year: 2022 Document Type: Article Affiliation country: S41598-022-11053-7