Scheme of the combined cleavage and purification strategy. [A] Procedure for proteins having a natural DAPase stop point. [B] Procedure for proteins with an introduced glutamine DAPase stop point.
Removal of the tag from 6xHis-tagged human tumor necrosis factor α (hTNFα) using the TAGZyme system. 1: Purified 6xHis-tagged hTNFα. 2: After incubation for 10 minutes with DAPase and Qcyclase enzymes at 37°C. 3: After incubation for 20 minutes with DAPase and Qcyclase enzymes at 37°C. 4: After incubation for 30 minutes with DAPase and Qcyclase enzymes at 37°C. 5: After completion of the reaction detagged, pyroglutamyl-extended hTNFα was recovered by subtractive IMAC, subjected to pGAPase digestion, and mature hTNFα was recovered in the flow-through fraction of a second round of subtractive IMAC. 6: An aliquot of the processed protein was incubated with excess DAPase (0.125 U/mg hTNFα) for 2 hours in order to analyze the efficiency of the pGAPase-catalyzed removal of pyroglutamate. The two subunits of His-tagged DAPase enzyme are visible. All samples were subjected to SDS-PAGE and the gel stained by Coomassie Blue. M: markers.
Schematic summary of the overall cleavage strategy using TAGZyme enzymes. [A] DAPase enzyme cleavage of a N-terminal His tag from a protein containing a natural stop point to obtain the mature target protein. [B] Cleavage of an N-terminal His tag from a protein making use of a glutamine stop point. Following dipeptide cleavage by DAPase enzyme, an N-terminal glutamine residue is converted to pyroglutamate that in turn is removed by pGAPase enzyme action.