Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

Abstract

This study systematically evaluated Escherichia coli (E. coli) expression systems for the production of a humanized single-chain variable fragment targeting interleukin-17A (hScFv-IL-17A). A wild-type hScFv (hScFv-IL-17A-WT), derived from Secukinumab, was compared with a C97S mutant (hScFv-IL-17A-C97S) in which the unpaired cysteine residue in the light-chain complementarity-determining region 3 (CDR3) was substituted with serine. The primary objectives were to identify an optimal expression system for soluble protein production and to assess the functional consequences of the C97S mutation. Protein solubility, yield, and antigen-binding properties were evaluated across expression on three E. coli strains: Origami B (DE3), SHuffle, and BL21 (DE3) incorporating the DisCoTune system. Analyses were performed using SDS-PAGE, Western blotting, enzyme-linked immunosorbent assay (ELISA), and biolayer interferometry (BLI). Among the systems tested, BL21 (DE3) with DisCoTune yielded the highest levels of soluble expression for both variants. Although the C97S mutation enhanced soluble protein yield in this system, it significantly impaired functional activity. The hScFv-IL-17A-WT exhibited a strong binding affinity (K_D = 3.64 × 10⁻⁸ M), whereas the hScFv-IL-17A-C97S demonstrated an approximately 10-fold reduction in affinity. Competitive ELISA further confirmed that both variants recognized the same epitope as a neutralizing monoclonal antibody. Structural modeling predicted that the C97S substitution imposed a moderate stability penalty without inducing major conformational alterations, consistent with the observed reduction in binding affinity. Collectively, these findings indicate that while the C97S mutation improves soluble expression, it compromises biological activity. The hScFv-IL-17A-WT produced in BL21 (DE3) using the DisCoTune system represents the most promising candidate for subsequent therapeutic development.