Utilization of Receptor-Binding Domain of SARS-CoV-2 Spike Protein Expressed in Escherichia coli for the Development of Neutralizing Antibody Assay

Abstract

The ongoing COVID-19 pandemic has resulted from widespread infection by the SARS-CoV-2 virus. As new variants of concern continue to emerge, understanding the correlation between the level of neutralizing antibodies (NAb) and clinical protection from SAR-CoV-2 infection could be critical in planning the next steps in COVID-19 vaccine programs. This study explored the potential usefulness of E. coli as an alternative expression system that can be used to produce a SARS-CoV-2 receptor-binding domain (RBD) for the development of an affordable and flexible NAb detection assay. We expressed the RBD of Beta, Delta, and Omicron variants in the E. coli BL21(DE3) strain and purified them from whole bacterial cells using His-tag-mediated affinity chromatography and urea-assisted refolding. Next, we conducted a head-to-head comparison of the binding activity of our E. coli-produced RBD (E-RBD) with commercial HEK293-produced RBD (H-RBD). The results of a direct binding assay revealed E-RBD and H-RBD binding with ACE2-hFc in similar signal strengths. Furthermore, in the NAb detection assay, % inhibition obtained from both E-RBD and H-RBD demonstrated comparable results in all the investigated assays, suggesting that non-glycosylated RBD produced from E. coli may offer a cost-effective alternative to the use of more expensive glycosylated RBD produced from human cells in the development of such an assay.

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model

“…Expression of SARS-CoV-2-RBD in E. coli could thus be advantageous from a production cost and scalability viewpoint [ 21 ]. However, owing to its four disulfide bonds, E. coli expression of SARS-CoV-2-RBD using standard protocols results in insoluble, non-native RBDs with aberrant disulfide bonds [ 22 , 23 ]. The reducing environment of the E. coli cytoplasm and the lack of molecular machinery to reshuffle non-native disulfide bonds into native ones make it unsuitable for producing multi-disulfide-bonded proteins [ 24 ].…”

“…Reversibility was assessed by measuring the spectrum after cooling the sample to 25 °C. The secondary structure content was calculated using BeStSel [ 22 ]. Tryptophan fluorescence: Trp fluorescence spectra with λex at 295 nm, respectively, were measured on an FP-8500 spectrofluorometer (JASCO, Tokyo, Japan) using a quartz cuvette with a 3 mm optical path length.…”

“…Cell surface CD markers on splenocytes extracted on the 147th day after the last dose were investigated by flow cytometry. Experimental samples (single mice from each group) were prepared according to our reported protocol [ 21 , 22 , 23 , 24 ]. In short, single-cell suspensions of mice splenocytes were prepared in a FACS (Fluorescence Activated cell Sorter) buffer (PBS supplemented with 2% FBS (Fetal Bovine Serum), 1 mM EDTA, and 0.1% sodium azide).…”

Functional Expression of the Recombinant Spike Receptor Binding Domain of SARS-CoV-2 Omicron in the Periplasm of Escherichia coli

“…Despite these merits, an E. coli expression system is not preferred in the production of a recombinant spike RBD, with four disulfide bonds [ 18 ] that are not correctly folded in the cytoplasm of E. coli, which is maintained in a reduced condition. Some researchers have tried to solve the folding problem by refolding the RBD [ 19 , 20 , 21 , 22 , 23 ], but the biochemical properties of the refolded RBD were quite different from those of the RBDs expressed in insect or mammalian cell systems [ 22 ]. The Borgstahl lab reported a soluble expression method of the recombinant RBD in the cytoplasm of E. coli using the enzyme (Evr1p and PDI)-assisted co-expression system CyDisCo [ 24 , 25 ].…”

Expression and purification of the receptor‐binding domain of SARS‐CoV‐2 spike protein in mammalian cells for immunological assays

“…Over the years, several heterologous expression systems have been developed to produce recombinant RBD. It can be expressed in bacteria, purified from inclusion bodies, and refolded [ 15 , 16 , 17 , 18 ]. However, bacterially produced RBD has some usage limits due to the lack of functionally relevant glycosylation, or heterotrimeric complex formation.…”