Improved Refolding of a Human IgG1 Fc (CH2-CH3) Scaffold from Its Inclusion Body in E. coli by Alkaline Solubilization

• Published in: Biological & pharmaceutical bulletin 2022/03/01, Vol.45(3), pp.284-291
• Main Authors: Ishikawa, Show, Ishikawa, Haruna, Sato, Atsushi
• Format: Article
• Language: English
• Subjects: alkaline solubilization; Circular dichroism; Dialysis; E coli; Escherichia coli; Fc receptors; Gel electrophoresis; human Fc (CH2-CH3); Immunoglobulin G; Immunostimulation; Inclusion bodies; inclusion body; pH effects; Polyacrylamide; Protein folding; Protein G; Proteins; refolding; Sodium lauryl sulfate; Solubilization; Spectroscopy
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b21-00796

Recently, we developed a platform strategy for hinge-deficient human immunoglobulin G1 (IgG1) Fc fusion as a non-immunostimulatory Fc fusion system. As a starting point to establish a promising approach for generating hinge-deficient Fc fusion proteins in Escherichia (E.) coli, we selected a CH2-CH3 scaffold as a model protein for evaluation. Recombinant CH2-CH3, expressed as inclusion bodies, was solubilized with various denaturants (urea, sarkosyl, sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Triton X-100) in neutral (phosphate-buffered saline (PBS), pH 8) or alkaline (50 or 500 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 11) buffer at 25 °C. Similar to the authentic CH2-CH3 produced in Chinese hamster ovary (CHO) cells, all denaturants, except urea in CAPS buffer but not in PBS, were found to elicit the dimer formation of solubilized CH2-CH3 on SDS-polyacrylamide gel electrophoresis (PAGE). After dialysis with PBS, sarkosyl-soluble CH2-CH3 inclusion bodies were successfully purified using protein G-Sepharose, indicating their successful refolding. Compared to the purified CH2-CH3 from its sarkosyl-soluble inclusion bodies in neutral buffer, that in 500 mM CAPS alkaline buffer revealed substantial structure-related similarities, such as secondary structures and thermal stabilities, as measured by circular dichroism spectroscopy, to authentic CH2-CH3. Native PAGE analysis also supported the above data. Therefore, solubilization at alkaline pH is an essential factor that promotes the refolding of CH2-CH3. Dimer formation of CH2-CH3 on SDS-PAGE may act as a surrogate marker for its protein refolding status. Our observations may provide important hints toward downstream processing of Fc-fusion production in E. coli.