A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

Brindha, Subbaian; Kuroda, Yutaka

doi:10.3390/ijms23031703

Cited by 15 publications

(14 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We start off by running simulations of RBD with or without distance constraints between native cysteine pairs, which mimic the effect of disulfide bonds (see Methods). Although our simulation force field is not currently equipped to model RBD’s N-linked glycans, we note that these glycans have been primarily linked to spike protein expression and cellular processing [51], and their absence does not appear to alter RBD structure, ACE2-binding, or immunogenicity [52]; likewise we do not expect their omission from simulations to drastically affect our findings.…”

Section: Resultsmentioning

confidence: 99%

Cotranslational formation of disulfides guides folding of the SARS COV-2 receptor binding domain

Bitran

Park

Serebryany

et al. 2022

Preprint

View full text Add to dashboard Cite

Many secreted proteins contain multiple disulfide bonds. How disulfide formation is coupled to protein folding in the cell remains poorly understood at the molecular level. Here, we combine experiment and simulation to address this question as it pertains to the SARS-CoV-2 receptor binding domain (RBD). We show that, whereas RBD can refold reversibly when its disulfides are intact, their disruption causes misfolding into a nonnative molten-globule state that is highly prone to aggregation and disulfide scrambling. Thus, non-equilibrium mechanisms are needed to ensure disulfides form prior to folding in vivo. Our simulations suggest that co-translational folding may accomplish this, as native disulfide pairs are predicted to form with high probability at intermediate lengths, ultimately committing the RBD to its metastable native state and circumventing nonnative intermediates. This detailed molecular picture of the RBD folding landscape may shed light on SARS-CoV-2 pathology and molecular constraints governing SARS-CoV-2 evolution.

show abstract

Section: Resultsmentioning

confidence: 99%

Cotranslational formation of disulfides guides folding of the SARS COV-2 receptor binding domain

Bitran

Park

Serebryany

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…RBD has also been expressed fused to a solubility-enhancing peptide (SEP) tag containing nine arginine residues, resulting in an enhancement in the accumulation of soluble RBD. This RBD version was produced at yields of up to 2 mg/L and recognized the ACE2 receptor and induced antibodies able to interact with a mammalian made S1 protein [ 33 ]. Another case is the report by McGuire et al [ 34 ], in which fusion proteins were designed based on the thermophilic family of nine carbohydrate-binding modules (CBM9) as an N-terminal carrier protein and affinity tag and different S protein segments.…”

Section: Discussionmentioning

confidence: 99%

Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2

et al. 2022

View full text Add to dashboard Cite

Most of the current SARS-CoV-2 vaccines are based on parenteral immunization targeting the S protein. Although protective, such vaccines could be optimized by inducing effective immune responses (neutralizing IgA responses) at the mucosal surfaces, allowing them to block the virus at the earliest stage of the infectious cycle. Herein a recombinant chimeric antigen called LTB-RBD is described, which comprises the B subunit of the heat-labile enterotoxin from E. coli and a segment of the RBD from SARS-CoV-2 (aa 439-504, carrying B and T cell epitopes) from the Wuhan sequence and the variant of concern (VOC)—delta. Since LTB is a mucosal adjuvant, targeting the GM1 receptor at the surface and facilitating antigen translocation to the submucosa, this candidate will help in designing mucosal vaccines (i.e., oral or intranasal formulations). LTB-RBD was produced in E. coli and purified to homogeneity by IMAC and IMAC-anionic exchange chromatography. The yields in terms of pure LTB-RBD were 1.2 mg per liter of culture for the Wuhan sequence and 3.5 mg per liter for the delta variant. The E. coli-made LTB-RBD induced seric IgG responses and IgA responses in the mouth and feces of mice when subcutaneously administered and intestinal and mouth IgA responses when administered nasally. The expression and purification protocols developed for LTB-RBD constitute a robust system to produce vaccine candidates against SARS-CoV-2 and its variants, offering a low-cost production system with no tags and with ease of adaptation to new variants. The E. coli-made LTB-RBD will be the basis for developing mucosal vaccine candidates capable of inducing sterilizing immunity against SARS-CoV-2.

show abstract

“…ELISA was carried out as previously described [17]. In short, anti-D3ED3 IgG levels were evaluated using native D3ED3 (2.5 μg/mL in PBS) as coating antigens on 96-well microtiter plates (TPP microtiter plates, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

The biophysical nature and not only the size of protein aggregates determines the strength of the immune response against dengue ED3

Kibria

Brindha

Kuroda

2022

Preprint

Self Cite

View full text Add to dashboard Cite

Here we used domain 3 of dengue virus serotype 3 envelope protein (D3ED3), a natively folded globular low-immunogenicity protein, to ask whether the biophysical nature of amorphous aggregates can affect immunogenicity. We prepared amorphous oligomers in five distinct ways. One oligomer type was produced using our SCP tag (Solubility Controlling Peptide) made of 5 Isoleucines (C5I). The others were prepared by miss-shuffling the SS bonds (Ms), heating (Ht), stirring (St), and freeze-thaw (FT). Dynamic light scattering showed that all five formulations contained oligomers of approximately identical sizes with hydrodynamic radii (Rh) between 30 and 55 nm. Circular dichroism (cd) indicated that the secondary structure content of oligomers formed by stirring and freeze-thaw was essentially identical to that of the native monomeric D3ED3. The secondary structure content of the Ms showed moderate changes, whereas the C5I and heat-induced (Ht) oligomers exhibited a significant change. Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti-D3ED3 IgG titer. Ht, St, and FT were barely immunogenic, similar to the monomeric D3ED3. Cell surface CD marker analysis by flow cytometry confirmed that immunization with Ms generated a strong central and effector T-cell memory. This result adds a new dimension to earlier studies where the strength of the immune response was associated solely with the presence and sizes of the oligomers. It also suggests that controlled oligomerization can provide a new, adjuvant-free method for increasing a protein's immunogenicity, yielding a potentially powerful platform for protein-based vaccines.

show abstract

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

Cited by 15 publications

References 39 publications

Cotranslational formation of disulfides guides folding of the SARS COV-2 receptor binding domain

Cotranslational formation of disulfides guides folding of the SARS COV-2 receptor binding domain

Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2

The biophysical nature and not only the size of protein aggregates determines the strength of the immune response against dengue ED3

Contact Info

Product

Resources

About