Assembly of a functional immunoglobulin F_v fragment in Escherichia coli

An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the F_v fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The F_v fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant F_v fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the F_v fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the F_v fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.