Application of an IMAC cassette for the purification of N-terminally tagged proteins

This study documents the use of an IMAC cassette for the purification of N-terminally tagged recombinant proteins. The procedures are based on the application of the immobilised macrocyclic ligand, 1,4,7-triazacyclononance (tacn), with a set of new tags specifically designed to interact with the corresponding immobilised Cu2+- and Ni2+-tacn complexes yet with the propensity to be efficiently removed by diaminopeptidases. The macrocyclic ligand, tacn, displays very high metal ion stability constants, whilst the bound metal ion retains the availability of co-ordination sites for interaction with the N-terminally tagged protein. These studies demonstrate that these new tags exhibit higher affinity and thus improved selectivity with both the immobilised Ni2+- and the Cu2+-tacn complexes compared to control tags, such as [His]6. This study thus documents an alternative IMAC platform for the highly selective, efficient and general purification of recombinant proteins.

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