Secretion of scFv Antibody Fragments

Antibodies consist of two functional parts, the antigen binding site, a heterodimer composed of the V_H and V_L domains, and the Fc part, which facilitates in vivo the effector functions and stability of the antibody. Recombinant antibodies were first successfully expressed by secretion from mammalian cells, whereas attempts to express whole antibodies in Escherichia coli remained unsuccessful. Later, the relatively small antigen binding portion of an antibody were functionally expressed as Fab or single-chain Fv (scFv) fragments in bacteria (1 ,2 ). Although intracellular expression of scFv fragments as inclusion bodies has been reported, most scFv antibodies are expressed in E. coli by secretion into the periplasmic space. By secretion across the cytoplasmic membrane, the heavy- and light-chain domains can fold and assemble properly, and intra- and interdomain disulfide bonds are formed (3 ,4 ). Single-chain antibodies purified from the periplasmic space of E. coli are primarily monomeric and, in most cases, functional. Although respectable yields of more than 200 mg/L of scFv fragment have been reported from large-scale E. coli fermentations (5 ), the yields obtained from small-scale expression cultures utilizing shake flasks are typically below 1 mg/L (4 ). Fermentation approaches gave rise to much higher yields, but these require thorough optimization and expensive equipment.