Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies

Recombinant single-chain antibodies (scFvs) that are expressed in the cytop lasm of cells are of considerable biotechnological and therapeutic potentia l. However, the reducing environment of the cytoplasm inhibits the formatio n of the intradomain disulfide bonds that are essential for correct folding and functionality of these antibody fragments. Thus, scFvs expressed in th e cytoplasm are mostly insoluble and inactive. Here, we describe a general approach for stabilizing scFvs for efficient fu nctional expression in the cell cytoplasm in a soluble, active form. The sc Fvs are expressed as C-terminal fusions with the Escherichia coli maltose-b inding protein (MBP). We tested a large panel of scFvs that were derived fr om hybridomas and from murine and human scFv phage display and expression l ibraries by comparing their stability and functionality as un-fused versus MBP fused proteins. We found that MBP fused scFvs are expressed at high lev els in the cytoplasm of E. coli as soluble and active proteins regardless o f the redox state of the bacterial cytoplasm. In contrast, most un-fused sc Fvs can be produced (to much lower levels) in a functional form only when e xpressed in trxB(-) but not in trxB(+) E. coli cells. We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that th ey perform more efficiently in vivo as cytoplasmic intrabodies in E. coli. Thus, MBP seems to function as a molecular chaperone that promotes the solu bility and stability of scFvs that are fused to it. (C) 2001 Academic Press .