A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants

Despite the well-known crucial role of intradomain disulfide bridges for im munoglobulin folding and stability, the single-chain variable fragment of t he anti-viral antibody F8 is functionally expressed when targeted to the re ducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia col i. A gel shift assay revealed that the single-chain variable fragment (scFv ) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl g roups. Guanidinium chloride denaturation/renaturation studies indicated tha t refolding occurs even in a reducing environment, producing a functional m olecule with the same spectral properties of the native scFv(Fs). Taken tog ether, these results suggest that folding and functionality of this antibod y fragment are not prevented in a reducing environment. This antibody fragm ent could therefore represent a suitable framework for engineering recombin ant antibodies to be targeted to the cytoplasm.