Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: Automation of batch adsorption measurements with tagged recombinant proteins

Changed loading conditions and lysate composition improve the purity of tagged recombinant proteins with tacn‐based IMAC adsorbents

“…A similar result was achieved for the Cu 2+ -dtnpSepharose™ 6 FF resin (data not shown). These findings are consistent with other studies related to IMAC resin screening [33], which have demonstrated that the Cu 2+ -IMAC Sepharose™ 6 FF resin has slower binding kinetics and lower protein selectivity as measured from the occupancy θ-value [34], making this adsorbent less efficient in terms of economic performance per cycle than the Cu 2+ -tacn-Sepharose™ 6 FF or dtnp-Sepharose™ 6 FF adsorbents.…”

“…As evident from these results, this simple pre-treatment increased the effective loading capacity for Cu 2+ -tacn-and Cu 2+ -dtnpSepharose™ 6 FF resins by at least 2-fold. These findings indicate that despite similarities in bulk Cu 2+ ion content for the three adsorbents, differences in binding capacities of the Cu 2+ -tacn-and Cu 2+ -dtnp-Sepharose™ 6 FF resins compared to the Cu 2+ -IMAC Sepharose™ 6 FF resin could be due to at least two effects, namely, surface chemistry differences between the tacn-related IMAC resins compared to the Cu 2+ -IMAC Sepharose™ 6 FF resin, resulting in different effective binding site θ occupancies [33,34]; and secondly due to different extents of binding of specific subsets of metal-binding metallophores or other nonspecific weak organic chelators, such as dicarboxylic acids, that are produced [37,38] [19][20]. In contrast, the Cu 2+ -tacn-and Cu 2+ -dtnp-Sepharose™ 6 FF adsorbents can be used for at least five cycles before the binding capacity fell below the threshold value of >50%, presumably due to the build-up of non-proteinaceous components made by the E. coli cells, stressed as a consequence of expression at higher levels of tagged heterologous proteins.…”

Separation and Characterization of Angiotensin I Converting Enzyme (ACE) Inhibitory Peptides from Saurida elongata Proteins Hydrolysate by IMAC-Ni2+

“…In the mid-1970s, Porath et al first described immobilized metal ion affinity chromatography (IMAC) [11]. Since that time, proteins of interest have been enriched through IMAC [12,13,14,15,16]. Nickel ion immobilized on solid supports have been widely used in IMAC [17,18].…”

Bi-Nuclear Metal Complexes of 2,6-Bis(1,4,7-triazacyclonon-1-yl-methylene)pyridine with Zinc(II), Copper(II), and Nickel(II)

“…As the pitch of an a-helix in a polypeptide or protein is 5.4 Å and for a b-sheet ,6 Å , these macromolecular metal ion complexes potentially have the ability to strongly bind to two histidine residues arranged either within the sequences of peptide tags such that a repeated i þ 4i hydrogen bonding motif occurs, typical of a right handed a-helix, or alternatively as a repeated C a i -C a iþ2 motif typical of b-strand structures. Experimental studies with recombinant proteins containing N-terminal tags with several histidine residues organised in such structural and conformational arrangements have confirmed [27][28][29] that strong binding does in fact occur, resulting in the efficient capture and purification of this class of tagged recombinant proteins by IMAC procedures based on the use of chromatographic resins derived from the immobilisation of these Cu II and Ni II complexes of bis(tacn) ligands. Interesting the corresponding immobilised Zn II complexes show very much lower binding affinities with the same tagged proteins, an observation which is consistent with the shorter distances between the two Zn II ions (Zn-Zn distance of ,3.38 Å ) as observed in this current investigation, indicating that only one histidine (rather than two) within the tag would be able to stochastically bind co-ordinatively to the immobilised Zn II complex of the IMAC adsorbent.…”