Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8360605 | Protein Expression and Purification | 2014 | 7 Pages |
Abstract
A new imidazolo- and thiol-containing ligand is prepared and characterized with the intent to serve as surrogate and mimic for the canonical ligands imidazole and hexa-histidinyl peptide (his-6) in immobilized metal affinity chromatography (IMAC) [1], [2] systems. The affinity of this ligand: 1,8 bis (N1,N1â²)imidazolo-octa-(3,6)-dithiane or bImOdS, to Ni-nitrilotriacetic acid (Ni-NTA) complex in solution is measured and compared with that of imidazole and his-6 peptide via isothermal titration calorimetry (ITC). In addition, bImOdS is compared with his-6 binding to the solid-state matrix of Ni-NTA-charged agarose beads, as employed routinely in IMAC. Results reported here include the following: (1) two imidazole moieties bind within a single Ni-NTA complex, while bImOds, being an imidazolo dimer, binds with 1:1, and his-6 peptide binds with 1:3 stoichiometry. (2) Enthalpies of reaction for imidazole and his-6 peptide are reported - these can be utilized to predict changes in affinity in IMAC systems with temperature, should protein unfolding/refolding steps in purification be desired at alternate temperatures. (3) Metal analyses of the Ni-NTA agarose beads suggests that â¼2/3 of the nickel is present in low-affinity sites, which will complicate protein separations at high protein-concentration loading. An improved procedure for subtracting ligand dilution heats from ITC analyses is presented in an Appendix.
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Authors
William R. Kirk,