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

Abstract

A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD’s eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell’s receptor, with a binding constant of 7.9 × 10−9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.

Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis

“…The need to obtain a glycosylated form of RBD is currently debatable. On the one hand, a fairly large number of studies have been published on the E. coli expressed RBD, which not only made it possible to obtain effective antibodies against the SARS-CoV-2 virus but also demonstrated its direct interaction with ACE2 with an efficiency close to the deglycosylated product (see, for example, [ 31 , 52 , 53 ]). It has also been shown that de-glycosylation of recombinant RBD samples expressed in mammalian cells does not significantly reduce the effectiveness of their interaction with ACE2 [ 54 ].…”

<i>E. coli</i> production of a multi-disulfide bonded SARS-CoV-2 Omicron BA.5 RBD exhibiting native-like biochemical and biophysical properties

“…The synthesized gene was inserted into pET15b at the NdeI/BamH1 with ampicillin resistance ( Supplementary Figure S1a ). The RBD was expressed in E. coli T7 SHuffle cells and purified from inclusion bodies as explained elsewhere [ 18 ]. The RBD proteins tagged by 6-Histidines were purified using denaturing open nickel-nitrilotriacetic acid (Ni-NTA) (Wako, Japan) chromatography.…”

“…The measurements were carried out under the condition of 10 mM Hepes buffer (pH 6.5) at a protein concentration of 0.3 mg/mL. The spectroscopic measurements: Far-UV circular dichroism (CD), Tryptophan fluorescence, Dynamic light scattering (DLS), and Static light scattering (SLS) were carried out, in essence, as reported previously [ 18 ].…”

“…We observed a full oxidation state at 72h as monitored by analytical RP-HPLC (Supplementary Figure S2b). After RP-HPLC purification, the elution profile showed a single sharp peak (Figure 1a), indicating that the purified RBD formed a single and most likely native species of disulfide bond pairing [18,[25][26][27]. The elution time of the fully oxidized and reduced RBD differed by about 2 minutes (Supplementary Figure S3).…”

A multi‐tier computational screening framework to effectively search the mutational space of SARS‐CoV‐2 receptor binding motif to identify mutants with enhanced ACE2 binding abilities

“…Also, spike contains many immunogenic hotspots. As a result, it is the prime target for vaccine and neutralizing antibody development [13][14][15][16][17][18][19].…”