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10.1016/j.jbiotec.2020.12.005

http://scihub22266oqcxt.onion/10.1016/j.jbiotec.2020.12.005
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33301853!7720734!33301853
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suck abstract from ncbi


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pmid33301853      J+Biotechnol 2021 ; 326 (ä): 21-27
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  • Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells #MMPMID33301853
  • Stuible M; Gervais C; Lord-Dufour S; Perret S; L'Abbe D; Schrag J; St-Laurent G; Durocher Y
  • J Biotechnol 2021[Jan]; 326 (ä): 21-27 PMID33301853show ga
  • Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHO(BRI/55E1) cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.
  • |*Protein Domains[MESH]
  • |*Recombinant Proteins[MESH]
  • |Animals[MESH]
  • |CHO Cells[MESH]
  • |Cricetulus[MESH]
  • |Gene Expression[MESH]
  • |HEK293 Cells[MESH]
  • |Humans[MESH]
  • |SARS-CoV-2[MESH]
  • |Spike Glycoprotein, Coronavirus/*genetics[MESH]


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