Co-Expression of Molecular Chaperones to Enhance Soluble aPD-L1 scFv Production in Escherichia coli BL21(DE3)

Abstract

Production of commercial antibodies commonly relies on mammalian expression platforms; however, these are associated with high production costs and technical challenges during large-scale cultivation. In this study, we aimed to develop an Escherichia coli-based platform for the production of an anti-PD-L1 single-chain variable fragment, hereafter aPD-L1 scFv. Expression of aPD-L1 scFv in E. coli BL21(DE3) resulted in extensive inclusion body formation regardless of the expression temperatures at 16 °C, 30 °C, and 37 °C, with approximately 84–93% accumulating in the insoluble fraction. To improve its solubility, co-expression with several molecular chaperones, namely DnaK-DnaJ-GrpE and GroES-GroEL, was investigated. Optimization of L-arabinose concentrations as an inducer for DnaK-DnaJ-GrpE expression demonstrated that induction at 0.15 mg/mL increased aPD-L1 scFv solubility from 5.13% to 23.97%, although the difference was not statistically significant. Moreover, His-tag purification yielded 14.78 µg/mL and 18.62 µg/mL without and with molecular chaperone co-expression, respectively. The binding activity of purified aPD-L1 scFv was confirmed by ELISA, which showed comparable binding signals following chaperone-assisted expression. This indicated that the functional activity was retained. Altogether, these findings suggest that molecular chaperone co-expression has the potential to increase the soluble production of functional aPD-L1 scFv in E. coli, supporting the feasibility of bacterial expression systems as a cost-effective platform for recombinant antibody fragment production.