Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from M(N+)-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 Cu(2+), Ni(2+), Zn(2+), Mn(2+), Co(2+), and Cr(3+), 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-deltaATPase-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-Ni(2+)-NTA). Investigations using the recombinant GST-deltaATPase-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-deltaATPase-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 Cu(2+) or Ni(2+), as IMAC systems.
ARC Special Research Centre for Green Chemistry, Monash University, Wellington Road, Victoria 3168, Australia.
Wei
W
Jiang
Mark
M
Prescott
Rodney J
RJ
Devenish
Leone
L
Spiccia
Milton T W
MT
Hearn
D001709:Biotechnology / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D015801:Helminth Proteins / Q000235:genetics / Q000302:isolation & purification; D008670:Metals / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification
D015801:Helminth Proteins; D008670:Metals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins