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. 2022 Feb 1;23(3):1703.
doi: 10.3390/ijms23031703.

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

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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

Subbaian Brindha et al. Int J Mol Sci. .

Abstract

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell's receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.

Keywords: Escherichia coli expression; disulfide bond; fusion tag; immunogenicity; solubility.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Strategy for cloning of SARS-CoV-2-C9R into pET15b vector. (A) Schematics of the sequence location of RBD in the SARS-CoV-2 spike protein and pET15b expression vector of RBD-C9R. (B) Amino acid sequence of the target gene cassette. (C) Ribbon model of RBD-C9R: The picture was generated using Pymol [20] with the coordinate of the PDB ID (6M0J) [3]. The tag region, shown in green, was generated using Modeller [21]. (D) SDS PAGE expression analysis: A1-soluble fraction of RBD-C9R, A2-pellet of RBD-C9R, B1-soluble fraction of RBD, B2-pellet fraction of RBD. (E) RP-HPLC elution profile of recombinant RBD-C9R showing the presence of a single peak indicated by an arrow. (F) RP-HPLC elution profile of RBD showing the presence of multiple broad peaks shown by arrows.
Figure 2
Figure 2
Biophysical and spectroscopic characterization: All spectroscopic measurements were performed at a protein concentration of 0.3 mg/mL in 10mM Hepes buffer, pH 8.0. (A) Far-UV CD spectrum of RBD-C9R (200–260 nm). (B) The melting temperature (Tm) of RBD-C9R monitored by CD at 220 nm, the raw data are shown with open circles (ο). (C) Tryptophan fluorescence spectra of RBD-C9R, (D) Tryptophan fluorescence spectra of RBD. (E) ANS fluorescence spectra of RBD-C9R, (F) ANS fluorescence spectra of RBD, (G) Dynamic Light Scattering, and (H) Static Light Scattering. Line symbols are explained within the panels.
Figure 3
Figure 3
QCM analysis of the RBD-C9R binding with hACE2. Injection plot for RBD concentrations. RBD proteins concentration are indicated in panel.
Figure 4
Figure 4
Immunogenicity of RBD-C9R. (A) Immunization scheme: A group of four mice was immunized with 0.3 mg/mL of RBD-C9R in 10 mM tris buffer without adjuvant. Up to three doses were injected biweekly, and tail bleed (TB) samples were collected three days after injection for monitoring the IgG titer by ELISA. M-indicates the mice’s identity, and TB indicates the tail bleeding number (once a week). (B) RBD-C9R IgG titer: IgG detection by ELISA was performed using the tail bleeding (TB) sera. Each circle indicates the individual mice titer, and the bars show the average titer (calculated over all four mice). (C) IgG sub-class (IgG1, IgG2a) Determination: Tail bleeding 5 (TB-5) serum sample was used for ELISA. (D) Recognition of mammalian expressed spike protein by RBD-C9R antisera: The binding of TB5 serum was measured using mammalian expressed spike (S1) protein as coating antigen and RBD-C9R as coating antigen for comparison. The plates were titrated using the mice antisera raised against RBD-C9R. The binding specificity of the antisera was determined with plates coated with lysozyme, as a control. The bar symbols are explained within the panel. (E) Absorbance at 492 nm vs. the reciprocal of antisera dilution against native Spike (S1) protein. (F) Absorbance at 492 nm vs. the reciprocal of antisera dilution against RBD-C9R (legends are given in the panel).

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