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Expression and characterization of SARS-CoV-2 spike proteins.
Schaub, Jeffrey M; Chou, Chia-Wei; Kuo, Hung-Che; Javanmardi, Kamyab; Hsieh, Ching-Lin; Goldsmith, Jory; DiVenere, Andrea M; Le, Kevin C; Wrapp, Daniel; Byrne, Patrick O; Hjorth, Christy K; Johnson, Nicole V; Ludes-Meyers, John; Nguyen, Annalee W; Wang, Nianshuang; Lavinder, Jason J; Ippolito, Gregory C; Maynard, Jennifer A; McLellan, Jason S; Finkelstein, Ilya J.
  • Schaub JM; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Chou CW; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Kuo HC; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Javanmardi K; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Hsieh CL; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Goldsmith J; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • DiVenere AM; Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
  • Le KC; Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
  • Wrapp D; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Byrne PO; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Hjorth CK; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Johnson NV; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Ludes-Meyers J; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Nguyen AW; Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
  • Wang N; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Lavinder JJ; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • Ippolito GC; Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
  • Maynard JA; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
  • McLellan JS; Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, TX, USA.
  • Finkelstein IJ; Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
Nat Protoc ; 16(11): 5339-5356, 2021 11.
Article in English | MEDLINE | ID: covidwho-1454802
ABSTRACT
The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Gene Expression Regulation, Viral / Spike Glycoprotein, Coronavirus / SARS-CoV-2 Topics: Vaccines / Variants Limits: Animals / Humans Language: English Journal: Nat Protoc Year: 2021 Document Type: Article Affiliation country: S41596-021-00623-0

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Gene Expression Regulation, Viral / Spike Glycoprotein, Coronavirus / SARS-CoV-2 Topics: Vaccines / Variants Limits: Animals / Humans Language: English Journal: Nat Protoc Year: 2021 Document Type: Article Affiliation country: S41596-021-00623-0