These investigations were designed to improve capture efficiency and selectivity in the immobilized metal ion affinity chromatographic (IMAC) purification of tagged recombinant proteins expressed in Escherichia coli cells, utilizing an alternative and novel class of immobilized metal binding ligands. The impact of loading conditions and lysate composition on the IMAC purification of NT1A- or His6-tagged green fluorescent protein (GFP), using the ligands 1,4,7-triazacyclononane (tacn) and bis(1,4,7-triazacyclononyl)propane (dtnp), charged with Cu2+ ions, has thus been explored. These findings were compared to the performance of a commercial adsorbent, IMAC Sepharose™ 6 FF, similarly charged with Cu2+ ions. With the same loading, wash and elution protocols, the tacn- and dtnp-derived adsorbents showed higher selectivity in terms of removal of E. coli host cell proteins than the commercial adsorbent, while low molecular weight components in the crude lysate had a higher impact on the binding capacities of tacn- and dtnp-derived adsorbents. This effect of lysate composition could be reduced through osmotic shock treatment of the E. coli cells prior to lysis. Additionally, the protein-binding capacities of the tacn-based resins were enhanced by increasing their ligand densities. Because both the tacn- and the dtnp-derived IMAC adsorbents exhibit very high metal ion stability constants, under the chromatographic conditions examined, they could be used several times without re-charging with Cu2+ ions. The results of these studies thus expand the general application scope of tacn-based IMAC resins for use in the capture and purification of tagged recombinant proteins.
- Affinity tag
- Bis(1,4,7-triazacyclononyl)propane (dtnp)
- Immobilized metal ion affinity chromatography (IMAC)
- Protein purification
- 1,4,7-Triazacyclononane (tacn)
Zhang, C., Fredericks, D. P., Longford, D. H., Campi, E. M., Sawford, T. R., & Hearn, M. T. W. (2015). Changed loading conditions and lysate composition improve the purity of tagged recombinant proteins with tacn-based IMAC adsorbents. Biotechnology Journal, 10(3), 480-489. https://doi.org/10.1002/biot.201400463