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Escherichia coli recombinant expression of SARS-CoV-2 protein fragments McGuire, Bailey E.; Mela, Julia E.; Thompson, Vanessa C.; Cucksey, Logan R.; Stevens, Claire E.; McWhinnie, Ralph L.; Winkler, Dirk F. H.; Pelech, Steven, 1957-; Nano, Francis E.

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.

Item Metadata

Title
Escherichia coli recombinant expression of SARS-CoV-2 protein fragments
Creator
McGuire, Bailey E.; Mela, Julia E.; Thompson, Vanessa C.; Cucksey, Logan R.; Stevens, Claire E.; McWhinnie, Ralph L.; Winkler, Dirk F. H.; Pelech, Steven, 1957-; Nano, Francis E.
Publisher
BioMed Central
Date Issued
2022-02-05
Description
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.
Subject
SARS-CoV-2; COVID-19; Recombinant spike protein; Carbohydrate-binding module; CBM9; Escherichia coli
Genre
Article
Type
Text
Language
eng
Date Available
2022-03-01
Provider
Vancouver : University of British Columbia Library
Rights
Attribution 4.0 International (CC BY 4.0)
DOI
10.14288/1.0406669
URI
http://hdl.handle.net/2429/80893
Affiliation
Medicine, Faculty of; Non UBC; Medicine, Department of
Citation
Microbial Cell Factories. 2022 Feb 05;21(1):21
Publisher DOI
10.1186/s12934-022-01753-0
Peer Review Status
Reviewed
Scholarly Level
Faculty; Other
Copyright Holder
The Author(s)
Rights URI
http://creativecommons.org/licenses/by/4.0/
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Attribution 4.0 International (CC BY 4.0)