A new strategy for the on-column exopeptidase cleavage of poly-histidine tagged proteins

Abstract

This paper describes a new strategy, which aims to make on-column poly-histidine tag removal more useful in the production of recombinant proteins by improving the yield and efficiency of on-column exopeptidase cleavage. This involves improvement of the on-column cleavage condition by using imidazole concentrations in the range of 100–500 mM in the cleavage buffer. At 300 mM imidazole, maximum on-column cleavage yield (in excess of 99%) was achieved in 3 h of incubation. However, as a result of the increased imidazole concentration, this new strategy of on-column cleavage results in some residual uncleaved poly-histidine tagged proteins (∼0.1%) and the production of cleaved dipeptides, both of which need to be further removed in a subsequent step. A method involving the recirculation of recovered proteins and peptides through the immobilized metal affinity chromatography (IMAC) column (same-column recirculation) was found to be superior to subtractive IMAC for the purpose of contaminant clearance. Recovery of the detagged target proteins was achieved using 10 column volumes of recovery buffer, which had the effect of diluting the imidazole concentration to a suitably low level for contaminant removal by same-column recirculation. This strategy was also applicable at a higher adsorbent loading of 10 mg protein/mL adsorbent with an optimal ratio of 200 mU of DAPase per mg of adsorbed tagged maltose binding protein (MBP), giving a cleavage yield of 99.1% in 3 h. Finally, on-column cleavage conditions including the effect of protease concentration and incubation time on the new strategy have been investigated and comparisons are made for different tag removal strategies.