Generation of a histidine-tagged antibotulinum toxin antibody fragment in E-coli:: Effects of post-induction temperature on yield and IMAC binding-affinity

Recombinant E. coli clones expressing a 50-kDa poly-histidine tail tagged antibody fragment against botulinum toxin (bt-Fab) were initially screened for yield and binding affinity. One clone was selected for bioprocess development. The selected bt-fab Vector was induced by addition of IPTG and the protein was targeted to the periplasm by inclusion of a pelB leader sequence. A histidine, affinity ligand at the heavy chain C-terminus facilitated single-step purification by immobilized metal-affinity chromatography (IMAC). Notably, the effects of post-induction temperature on bt-fab expression and downstream purification were evaluated. Our results demonstrated that fermentation conditions interfered with purification on the IMAC column at 37 degrees C. Protease analysis by gelatin polyacrylamide gel electrophoresis (GPAGE) indicated the presence of a membrane-bound similar to 39 kDa protease activity shortly after induction. The appearance of the protease activity was inversely correlated with the bt-fab yield. The protease was purified and was shown to degrade bt-fab. A simple kinetic model was developed describing temporal regulation of protease and bt-fab degradation. partially degraded bt-fab was unrecoverable by IMAC, presumably due to the loss of the His, affinity ligand. The amount of purified bt-fab obtained per liter of fermentation broth was typically similar to 1 mg.