Microbial Cell Factories (Feb 2022)

Escherichia coli recombinant expression of SARS-CoV-2 protein fragments

  • Bailey E. McGuire,
  • Julia E. Mela,
  • Vanessa C. Thompson,
  • Logan R. Cucksey,
  • Claire E. Stevens,
  • Ralph L. McWhinnie,
  • Dirk F. H. Winkler,
  • Steven Pelech,
  • Francis E. Nano

DOI
https://doi.org/10.1186/s12934-022-01753-0
Journal volume & issue
Vol. 21, no. 1
pp. 1 – 13

Abstract

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Abstract We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline–threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540–588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

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