Separation of Hexahistidine Fusion Proteins With Immobilized Metal Ion Affinity Chromatographic (IMAC) Sorbents Derived From MN+-Tacn and Its Derivatives

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7-triazacyclononane (tacn), bis(1,4,7-triazacyclononyl) ethane (dtne) and bis( 1,4,7-triazacyclononyl) propane (dtnp) complexed with the borderline metal ions Cu2+, Ni2+, Zn2+, Mn2+, CO2+, and Cr3+, for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C-terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S-transferase-Saccharomyces cerevisiae mitochondrial ATP synthase delta-subunit (GST-delta ATPase-HiS(6)), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)-nitrilotriacetic acid (im-Ni2+-NTA). Investigations using the recombinant GST-delta ATPase-HiS(6) and recombinant S. japonicum glutathione S-transferase (GST) lacking a hexahistidine tag have confirmed that the C-terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST-delta ATPase-HiS(6) can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu2+ or Ni2+, as IMAC systems. Biotechnol. Bioeng. 2009;103: 747-756. (c) 2009 Wiley Periodicals, Inc.