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

Microbial Cell Factories

Logan R. Cucksey¹  Francis E. Nano¹  Bailey E. McGuire¹  Ralph L. McWhinnie¹  Julia E. Mela¹  Claire E. Stevens¹  Vanessa C. Thompson¹  Dirk F. H. Winkler²  Steven Pelech³ 

[1] Department of Biochemistry and Microbiology, University of Victoria, STN CSC, PO Box 1700, V8W 2Y2, Victoria, BC, Canada;Kinexus Bioinformatics Corporation, Vancouver, BC, Canada;Kinexus Bioinformatics Corporation, Vancouver, BC, Canada;Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada;

关键词: SARS-CoV-2;    COVID-19;    Recombinant spike protein;    Carbohydrate-binding module;    CBM9;    Escherichia coli;   

DOI  :  10.1186/s12934-022-01753-0

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY