Assembly of a Functional Immunoglobulin F v Fragment in Escherichia coli

• DOI: 10.1126/science.3285470
• Publication Date: 2006-10-05T17:21:15Z
• Authors: Arne Skerra; Andreas Plückthun
• Keywords: 0303 health sciences; 03 medical and health sciences; Myeloma Proteins; Phosphorylcholine; Genetic Vectors; Escherichia coli; Immunoglobulin Variable Region; Immunoglobulin Fragments; Recombinant Proteins; Plasmids; 3. Good health

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.