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

• Published in: Microbial cell factories Vol. 21; no. 1; p. 21
• Main Authors: McGuire, Bailey E, Mela, Julia E, Thompson, Vanessa C, Cucksey, Logan R, Stevens, Claire E, McWhinnie, Ralph L, Winkler, Dirk F H, Pelech, Steven, Nano, Francis E
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 05.02.2022; BioMed Central; BMC
• Subjects: Amino acids; Analysis; Antibodies; Antibodies, Viral - immunology; Antigen-Antibody Reactions; Antigenic determinants; Antigens; Binding; Carbohydrate-binding module; Carbohydrates; CBM9; Cellulose; Chromatography, High Pressure Liquid; Cloning; Control; Coronavirus Nucleocapsid Proteins - genetics; Coronavirus Nucleocapsid Proteins - metabolism; Coronaviruses; COVID-19; COVID-19 - pathology; COVID-19 - virology; COVID-19 vaccines; E coli; Enzymes; Epitopes; Escherichia coli; Escherichia coli - metabolism; Fragments; Fusion protein; Genomes; Humans; Identification and classification; Mass Spectrometry; Modules; Nucleocapsids; Peptides; Phosphoproteins - genetics; Phosphoproteins - metabolism; Polymerase chain reaction; Potassium; Proline; Proteins; Receptors, Cell Surface - genetics; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant proteins; Recombinant spike protein; SARS-CoV-2; SARS-CoV-2 - isolation & purification; SARS-CoV-2 - metabolism; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Spike Glycoprotein, Coronavirus - genetics; Spike Glycoprotein, Coronavirus - metabolism; Spike protein; Testing; Threonine; Viral antibodies; COVID-19; Carbohydrate-binding module; SARS-CoV-2; CBM9; Recombinant spike protein; Escherichia coli
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-022-01753-0

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.