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Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.
Johari, Yusuf B; Jaffé, Stephen R P; Scarrott, Joseph M; Johnson, Abayomi O; Mozzanino, Théo; Pohle, Thilo H; Maisuria, Sheetal; Bhayat-Cammack, Amina; Lambiase, Giulia; Brown, Adam J; Tee, Kang Lan; Jackson, Philip J; Wong, Tuck Seng; Dickman, Mark J; Sargur, Ravishankar B; James, David C.
  • Johari YB; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Jaffé SRP; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Scarrott JM; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Johnson AO; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Mozzanino T; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Pohle TH; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Maisuria S; Department of Immunology, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, UK.
  • Bhayat-Cammack A; Department of Immunology, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, UK.
  • Lambiase G; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Brown AJ; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Tee KL; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Jackson PJ; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Wong TS; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Dickman MJ; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
  • Sargur RB; Department of Immunology, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, UK.
  • James DC; Department of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, UK.
Biotechnol Bioeng ; 118(2): 1013-1021, 2021 02.
Article in English | MEDLINE | ID: covidwho-893204
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ABSTRACT
We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Spike Glycoprotein, Coronavirus / COVID-19 Testing / SARS-CoV-2 / COVID-19 Type of study: Cohort study / Diagnostic study / Observational study / Prognostic study Limits: Animals / Humans Language: English Journal: Biotechnol Bioeng Year: 2021 Document Type: Article Affiliation country: Bit.27615

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Spike Glycoprotein, Coronavirus / COVID-19 Testing / SARS-CoV-2 / COVID-19 Type of study: Cohort study / Diagnostic study / Observational study / Prognostic study Limits: Animals / Humans Language: English Journal: Biotechnol Bioeng Year: 2021 Document Type: Article Affiliation country: Bit.27615