An Optimized and Systematically Integrated Expression, Extraction, and Purification Strategy Enabling Efficient Production of TCR-Like Antibodies in Escherichia coli.

Efficient bacterial production of recombinant antibody fragments is critical for structural characterization and subsequent functional studies. In this study, a systematically optimized workflow was developed for the expression, extraction, and purification of soluble T-cell receptor (TCR)-like antibody fragments in Escherichia coli (E. coli). Two expression hosts, the K-12-derived HB2151 strain and the B-lineage BL21(DE3) pLysS strain, were comparatively evaluated. Although both strains supported recombinant expression, BL21(DE3) pLysS produced higher levels of soluble protein. However, conventional osmotic shock extraction resulted in limited protein recovery. To improve extraction efficiency, a combined lysis approach incorporating freeze-thaw cycles, lysozyme treatment, and sonication was applied, increasing total protein recovery in cell lysates to 149.2-178.4 mg/L of culture. Purification was carried out using Protein A affinity chromatography, and the purification conditions were further optimized to enhance protein recovery. Optimal binding was achieved using 20 mM sodium phosphate buffer (pH 6.8), while elution with 100 mM citric acid (pH 2.9) improved protein yield while maintaining protein stability during purification. Under these conditions, purified soluble TCR-like antibody fragments were obtained at final yields of 6.16-7.08 mg/L of culture. The purified proteins migrated at the expected molecular weight (~ 15 kDa) on SDS-PAGE and were specifically detected by dot blot analysis. Overall, this study provides a reproducible and scalable workflow for improving the expression, extraction, and purification of recombinant soluble TCR-like antibody fragments in E. coli.