బయోకెమిస్ట్రీ & మాలిక్యులర్ బయాలజీ జర్నల్ అందరికి ప్రవేశం

నైరూప్య

Fusion of Zif268 to the C-Terminus of Scfvs Promotes Expression of the Active Form in the Cytoplasm of Escherichia coli

Mieko Kato and Yoshiro Hanyu

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.