EGFR extracellular domain III expressed in Escherichia coli with SEP tag shows improved biophysical and functional properties and generate anti-sera inhibiting cancer cell growth

Biochem Biophys Res Commun. 2021 May 28;555:121-127. doi: 10.1016/j.bbrc.2021.03.102. Epub 2021 Apr 1.

Abstract

The epidermal growth factor receptor extracellular domain III (EGFR-ECDIII) protein is a promising target of anti-cancer research, and its production in Escherichia coli would thus represent significant benefits. However, despite its moderate size (19 kDa), the expression of EGFR-ECDIII in E.coli is hampered by the presence of multiple cysteines producing misfolded proteins with incorrect S-S bonds. In our study, we show that a short 12-residue solubility enhancing peptide (SEP) tag containing nine arginines (C9R) attached at the C-terminus of EGFR-ECDIII reduces the inclusion body formation and increases the final yield by six times (20 mg/L). EGFR-ECDIII-C9R purified from the soluble fraction eluted as a sharp single RP-HPLC peak, suggesting a single S-S bond pairing. Biophysical characterization using circular dichroism, fluorescence, and light scattering confirmed its native-like properties together with reversible thermal denaturation. The binding activity of EGFR-ECDIII-C9R to anti-EGFR-VHH7D12, a single-domain antibody with specific binding to the ECDIII, was assessed by sandwich ELISA. Further, we produced anti-EGFR-ECDIII-C9R antisera in mouse models and anti-sera inhibited A431 cancer cells' growth. These results demonstrate that the SEP tag enables the rapid production of the multiple disulfide-bonded EGFR-ECDIII in E. coli having native-like biophysical properties and producing neutralizing antibodies.

Keywords: Disulfide bond; Escherichia coli expression; Immunogenicity; Neutralizing antibody; Solubility.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents, Immunological / immunology
  • Antineoplastic Agents, Immunological / pharmacology*
  • Cell Line, Tumor
  • Chromatography, Reverse-Phase
  • Circular Dichroism
  • Enzyme-Linked Immunosorbent Assay
  • ErbB Receptors / genetics*
  • ErbB Receptors / immunology
  • Escherichia coli / genetics
  • Female
  • Humans
  • Immune Sera
  • Mice, Inbred ICR
  • Particle Size
  • Protein Conformation
  • Protein Domains
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / immunology*
  • Recombinant Proteins / isolation & purification
  • Solubility

Substances

  • Antineoplastic Agents, Immunological
  • Immune Sera
  • Recombinant Proteins
  • ErbB Receptors