Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells
To test whether the accumulation of cytoplasmically targeted recombinant antibodies could be improved by fusion to a cytoplasmic protein, we generated a series of single chain antibody-fusion proteins and assayed the levels of functional protein. Glutathione S-transferase (GST) from S. japonicum, coat protein (CP) from TMV, thioredoxin from tobacco (TRXt) or thioredoxin from E. coli (TRXe) was fused to the N-terminus of scFv24, a TMV specific single chain antibody. Accumulation of functional fusion proteins in the endoplasmic reticulum (ER) and plant cell cytoplasm was analysed by transient expression in tobacco leaves. ELISA analysis demonstrated that the fusion partners did not prevent the binding of scFv24 to TMV virions. However, accumulation of functional scFv24 was dependent on the fusion partner coupled to it. CP-scFv and GST-scFv fusion protein accumulation amounted to 1ng and 3ng per gram leaf material, respectively, whereas the thioredoxin fusion proteins were produced at low levels. Western blot and surface plasmon resonance analysis confirmed the integrity of the ER retained CP and GST fusion proteins. In the cytoplasm, only the CP fusion protein was detectable (1-5ng per gram leaf material) and levels of scFv24 alone or fused to the other three fusion partners were below the ELISA detection limit. Addition of a KDEL sequence to the C-terminus of the cytoplasmic CP fusion resulted in a 3 fold increase in protein accumulation indicating that an N-terminal CP and the C-terminal KDEL sequence are suitable elements to stabilize scFv antibodies in the cytoplasm.