Molecular chaperone effects on recombinant yield and binding characteristics of an ABA-specific scFv in Escherichia coli

作者：S.-L. Chen, Bin Shan, Yican Luo, Guodong Mo, Usman Rashid Malik, Lin‐Li Lv, Xinyu Yang, Qi Lu · 发表于：Frontiers in Bioengineering and Biotechnology · 年份：2025 · DOI：10.3389/fbioe.2025.1643833 · 被引用次数：2 · 研究领域：Monoclonal and Polyclonal Antibodies Research、Protein purification and stability、Transgenic Plants and Applications

Introduction Recombinant single‐chain variable fragments (scFvs) are promising antibody formats for cost‐effective and scalable production. However, their soluble expression in Escherichia coli is often limited by misfolding and aggregation, particularly for scFvs targeting small molecule haptens such as abscisic acid (ABA). To address this bottleneck, molecular chaperones can be co‐expressed to enhance folding efficiency and functional yield. Methods An ABA‐specific scFv was expressed in E. coli BL21(DE3) using five different chaperone plasmids (pG‐KJE8, pGro7, pKJE7, pG-Tf2, and pTf16). Soluble expression was quantified by His‐tag ELISA, and protein identity was confirmed by SDS‐PAGE and Western blot. Functional characterization included competitive ELISA for IC 50 and specificity, while secondary structure was analyzed by FT‐IR and circular dichroism spectroscopy. Results Trigger Factor (pTf16) significantly improved soluble scFv yield (19.65%) compared to the control (14.20%). The pKJE7 system achieved the highest sensitivity with the lowest IC 50 , whereas the pTf16 system provided superior specificity and a broader detection range by minimizing cross‐reactivity. Structural analysis revealed that pKJE7‐assisted scFv closely matched the predicted β‐sheet content, correlating with high sensitivity, while pTf16‐assisted scFv avoided non-native α-helices, supporting enhanced specificity. Circular dichroism further demonstrated that pKJE7‐ and pTf16‐assisted scFvs exhibited c...