Intrabody construction and expression. II. A synthetic catalytic Fv fragment

In general, proteins with structural disulfides cannot be expressed in the reducing environment of the cellular cytoplasm. To overcome this folding pr oblem, we have previously engineered stabilizing mutations, predicted from a consensus sequence analysis, into isolated immunoglobulin V-L domains. He re we show that such domains can be used as a framework in the construction of a functional heterodimeric Fv fragment, which was expressed solubly, wi th high yield in the cytoplasm of Escherichia coli. This designed catalytic intrabody, obtained from grafting the combining site of the esterolytic an tibody 17E8, is active in the oxidized and the reduced state. Its construct ion required no special features on the part of the immunoglobulin, no sing le-chain linker and introduced no non-natural sequence motifs. The potentia l to design intrabodies with the recognition sequences of arbitrary immunog lobulins opens novel opportunities for gene therapy, cell biology, metaboli c engineering and antibody biotechnology. (C) 1999 Academic Press.