High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida pl ants. The five scFv-encoding sequences were cloned in an optimized plant tr ansformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed p etunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fr agments accumulated to unexpectedly high levels in the cytosol of the prima ry transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and f unctional. Such scFv scaffolds provide new opportunities to design scFv fra gments for immunomodulation in the cytosol.