FUNCTIONAL ANTIBODY-PRODUCTION USING CELL-FREE TRANSLATION - EFFECTS OF PROTEIN DISULFIDE-ISOMERASE AND CHAPERONES

To create a rapid system to test the effect of sequence changes on rec ombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translatio n system. Functional antibodies with antigen-binding activity are obta ined only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulf ide isomerase (PDI) leads to a threefold increase in yield over that o btained in the presence of glutathione redox systems. DsbA had no such effect, indicating that disulfide shuffling, and not net formation, i s the crucial yield-limiting step. The addition of the molecular chape rones DnaK and DnaJ increased the amount of soluble protein but not th e amount of functional scFv, which appears to be limited entirely by c orrect disulfide formation. None of these factors significantly influe nced total protein synthesis. In the presence of PDI, chaperones, redu ced glutathione and oxidized glutathione, 50% of the scFv produced (ab out 8 mu g/ml in only 15 min) could be recovered from immobilized anti gen.