Enhancing Soluble Expression and Purification of a Bispecific Scfv Antibody in Escherichia Coli Using Chaperones Publisher



Alidoustishahraki Z ; Aminidehkordi A ; Esmaeil N ; Akbari V
Source: Research in Pharmaceutical Sciences Published:2026

Abstract

Background and purpose: The use of a bispecific antibody engineered as a tandem single-chain fragment variable antibody (scFv-BsAb) offers several advantages over full-length antibodies, including enhanced tissue penetration and the potential for production in bacterial cells. However, production of them in bacteria usually results in the formation of inclusion bodies. The current work aimed to facilitate proper folding and soluble expression of a recombinant scFv-BsAb targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) by co-expression with molecular chaperones. Experimental approach: Escherichia coli BL21(DE3) cells harboring pET28a-scFv-BsAb and pKJE7 plasmids were used as host cells for protein expression. The effect of different temperatures and IPTG concentrations on the expression of soluble protein with or without expression of chaperones was investigated by SDS-PAGE and densitometry analysis. Subsequently, the expressed protein was subjected to native purification using a Ni-NTA affinity column. Then, the binding ability of the scFv-BsAb to the receptors on peripheral blood mononuclear cells (PBMCs) was evaluated by flow cytometry. Findings/Results: Co-expression with chaperones slightly enhanced the expression of soluble scFv-BsAb. The optimal condition for soluble expression of protein is at a temperature of 30 °C and an IPTG concentration of 1 mM when co-expressed with chaperones. The final yield of protein in co-expression with chaperones was 2.9 mg protein per 1 L of culture. Conclusion and implications: Optimization of culture conditions and co-expression with molecular chaperones may be useful for the production of soluble and functionally bioactive scFv antibodies in E. coli. © 2026 Research in Pharmaceutical Sciences.