Abstract

The expression of functional scFvs in Escherichia coli cytoplasm at high yields remains a challenge, because the reducing environment of the cytoplasm inhibits disulfide bond formation, which is essential for efficient and appropriate folding of scFvs. Thus, to address this challenge, we aimed to develop a method for the efficient functional expression of scFvs in E. coli cytoplasm. The scFv against rabbit IgG (scFv(A10B)) fused with Zif268 at its C-terminus was expressed at high levels in the cytoplasm of E. coli in a soluble and active form. The reactivity Zif268-fused scFv against the antigen was identical to that of un-fused scFv(A10B). In contrast, un-fused scFv(A10B) can be produced in a functional form only when expressed in the oxidizing environment of the periplasmic space, which limits expression quantities. Fusion with maltose-binding protein (MBP) did not improve expression. We compared the productivity and functionality of un-fused and Zif268-fused proteins by using several scFvs derived from hybridomas and a chicken scFv phage display library. We found that Zif268-fused scFvs are expressed at a high level in the cytoplasm of E. coli as soluble and active proteins.