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Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from M N + ‐tacn and its derivatives
Author(s) -
Jiang Wei,
Prescott Mark,
Devenish Rodney J.,
Spiccia Leone,
Hearn Milton T.W.
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22302
Subject(s) - iminodiacetic acid , chemistry , nitrilotriacetic acid , metal ions in aqueous solution , chelation , metal , chromatography , affinity chromatography , ligand (biochemistry) , recombinant dna , biochemistry , enzyme , inorganic chemistry , organic chemistry , gene , receptor
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 δ‐subunit (GST‐δATPase‐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‐δATPase‐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‐δATPase‐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. Biotechnol. Bioeng. 2009;103: 747–756. © 2009 Wiley Periodicals, Inc.