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

Int J Mol Sci. 2022 Feb 1;23(3):1703. doi: 10.3390/ijms23031703.

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.

MeSH terms

  • Angiotensin-Converting Enzyme 2 / metabolism
  • Animals
  • Antibodies, Viral / blood*
  • Cloning, Molecular
  • Disulfides / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / growth & development*
  • Female
  • Humans
  • Immune Sera / metabolism
  • Immunization
  • Mice
  • Mice, Inbred ICR
  • Peptides / administration & dosage*
  • Peptides / genetics
  • Peptides / immunology
  • Protein Domains
  • Spike Glycoprotein, Coronavirus / chemistry*
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / immunology
  • Th2 Cells / metabolism

Substances

  • Antibodies, Viral
  • Disulfides
  • Immune Sera
  • Peptides
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2