Process intensification for the removal of poly-histidine fusion tags from recombinant proteins by an exopeptidase.
This study describes the use of a hexa-histidine tagged exopeptidase for the cleavage of hexa-histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On-column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on-column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post-load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post-load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa-histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa-histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on-column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on-column exopeptidase cleavage makes the integration of the poly-histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly-histidine tag removal can now be combined with the on-column tag cleavage operation.
Kuo
Wen-Hui K
WH
Dept. of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, U.K. whkk2@cam.ac.uk
Chase
Howard A
HA
D002846:Chromatography, Affinity; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D020689:Exopeptidases / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009842:Oligopeptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*
C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D020689:Exopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases