Stabilization of affinity-tagged recombinant protein during/after its production in a cell-free system using wheat-germ extract

We found that the affinity tag fused to the carboxyl (C-) terminal of a single-chain Fv (scFv) antibody was proteolytically degraded in a wheat germ cell-free protein synthesis system. The addition of two extra residues of glycine to the tail of the cMyc tag significantly increased the stability of the tag, suggesting that wheat endogenous carboxypeptidase(s) play a primary role in the C-terminal tag-specific degradation. In addition to the modification of the tag sequence, addition of diisopropyl fluorophosphate, which is known as an inhibitor of carboxypeptidases, prevented the cMyc tag sequence degradation. The effects of other protease inhibitors on the translation reaction and stability of the synthesized protein are also reported.