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Escherichia coli recombinant expression of SARS-CoV-2 protein fragments. (preprint)
biorxiv; 2021.
Preprint
in English
| bioRxiv | ID: ppzbmed-10.1101.2021.06.22.449540
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 used the thermophilic carbohydrate binding domain 9 (CBM9) module 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 rich 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 fusion proteins were expressed in E. coli at about 0.1 g/L, and could be purified with a single affinity binding step using inexpensive cellulose powder. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibody directed to the appropriate SARS-CoV-2 antigenic region. The largest CBM9 fusion protein incorporates a spike protein self-folding domain, and includes amino acids 540-588 of the spike protein. This conserved region is immediately C-terminal to the receptor binding domain, is widely recognized by human convalescent sera, and contains a putative protective epitope.
Full text:
Available
Collection:
Preprints
Database:
bioRxiv
Language:
English
Year:
2021
Document Type:
Preprint
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