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

Abstract

These investigations were designed to improve capture efficiency and selectivity in the immobilized metal ion affinity chromatographic (IMAC) purification of tagged recombinant proteins expressed in Escherichia coli cells, utilizing an alternative and novel class of immobilized metal binding ligands. The impact of loading conditions and lysate composition on the IMAC purification of NT1A- or His6 -tagged green fluorescent protein (GFP), using the ligands 1,4,7-triazacyclononane (tacn) and bis(1,4,7-triazacyclononyl)propane (dtnp), charged with Cu(2+) ions, has thus been explored. These findings were compared to the performance of a commercial adsorbent, IMAC Sepharose™ 6 FF, similarly charged with Cu(2+) ions. With the same loading, wash and elution protocols, the tacn- and dtnp-derived adsorbents showed higher selectivity in terms of removal of E. coli host cell proteins than the commercial adsorbent, while low molecular weight components in the crude lysate had a higher impact on the binding capacities of tacn- and dtnp-derived adsorbents. This effect of lysate composition could be reduced through osmotic shock treatment of the E. coli cells prior to lysis. Additionally, the protein-binding capacities of the tacn-based resins were enhanced by increasing their ligand densities. Because both the tacn- and the dtnp-derived IMAC adsorbents exhibit very high metal ion stability constants, under the chromatographic conditions examined, they could be used several times without re-charging with Cu(2+) ions. The results of these studies thus expand the general application scope of tacn-based IMAC resins for use in the capture and purification of tagged recombinant proteins.

Interactions between an amphipathic di‐histidine peptide and a metal affinity chromatographic resin derived from a bis(tacn)butane chelating ligand

“…Importantly, the adsorption isotherm analyses indicate that the binding behavior between the peptide (1) and im-M n+ -bis(tacn) but Sepharose CL-4B sorbents cannot be adequately described by a first order Langmuir isotherm involving a single binding energy and monolayer surface coverage. Consistent with other data for recombinant proteins that are N-terminally tagged with this and other histidine-rich peptides [25,27,31,32,47], the synthetic peptide had a higher binding affinity with the im-Cu 2+ -bis(tacn) but Sepharose CL-4B sorbent than with im-Ni 2+ -bis(tacn) but Sepharose CL-4B sorbent. The low binding observed with the im-Zn 2+bis(tacn) but -Sepharose CL-4B sorbent under all conditions suggested that all six coordination sites on the im-Zn 2+ ion are occupied.…”

“…These characteristics will result in heterogeneity in binding sites and account for divergence from simple Langmuirean isotherms. These characteristics would also account for the capture of acidic HCPs that lack histidine residues in their primary structure as well as why some histidine-containing HCPs, and even cell culture media derived proteins, are co-purified as sorbent-bound species with the target histidine-tagged recombinant protein generated by fermentation/cell culture methods [27][28][29]31,47]. As documented in these investigations, a parallel exists between the binding site heterogeneity shown by the im-M 2+bis(tacn) but -Sepharose CL-4B sorbents and the properties of the corresponding sorbents generated with the chelating ligands IDA and NTA, where the capture of specific HCPs and non-proteinaceous metallophores are also observed.…”

“…To date, only a few studies have investigated the biophysical nature of the interaction between different peptide tag motifs and borderline metal ions chelated to immob‐ilized tacn‐related ligands. Although significant purification factors can be achieved with bacterial fermentates and mammalian cell culture supernatants for recombinant proteins containing N‐ or C‐terminal polyhistidine tags using IMAC methods, a variety of specific host cell proteins (HCPs), non‐proteinaceous metallophores, and non‐specific chelating secondary metabolites derived from the citric acid cycle also co‐purify, leading to contamination of the product . Although some of the HCPs contain histidine‐rich motifs, other HCPs, such as the highly basic helicase Rh1E , do not contain any histidine residues within their amino acid sequences, but are also captured by IMAC sorbents.…”

Using iSUSTAIN™ to validate the chemical attributes of different approaches to the synthesis of tacn and bridged (bis)tacn ligands

“…30,39,40 We have recently reported the use of both tacn (1) and (bis)tacn (2) as ligands for the more benign and efficient purification of tagged recombinant proteins using IMAC. 41,42 Conceptually, the synthesis of tacn ( 1) and (bis)tacn compounds (2) can be achieved in several ways, using different starting materials, reagents and conditions. This makes the set of reactions an ideal target for analysis using the iSUSTAIN™ toolkit.…”