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Studies in immobilized metal ion affinity chromatography of proteins

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posted on 16.01.2017 by Widakowich, Gabriel Stephan Alexander Leone
Immobilized metal ion affinity chromatography (IMAC) is a powerful tool for purification of proteins from complex mixtures. The technique is based on the interaction of electron donor groups on the surface of proteins with immobilized metal chelates. Currently, many IMAC systems employ borderline metal ions such as Ni2+, Cu2+ and Co2+, according to the classification of Pearson, chelated to linear ligands like iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA). The stability constants of Ni2+ and Cu2+ chelates with IDA and NTA are relatively low (log β = 8-13). As a result, metal ions can leak during the chromatographic process. This is undesirable due to the toxicity of borderline metal ions. The aim of this thesis was to contribute to the development of IMAC systems without leakage of borderline metal ions. Two strategies were employed – using borderline metal ions but minimizing their leakage, and employing the more benign metal ion Ca2+. Strategy 1 was pursued through studies of IMAC systems composed of the ligands 1,4,7-triazacyclononane (tacn) and bis(1,4,7-triazacyclonon-1-yl)propane (propyl-bis-tacn), forming stable metal complexes with Ni2+ and Cu2+ (log β ~ 16), and the affinity tag NT1A. To facilitate sequential purification, the NT1A tag was fused in sequence with the putative hard metal binding tag HIT2. Two studies were performed to test the suitability of the affinity tags NT1A and HIT2. A. The arrangement of the NT1A and HIT2 tags with respect to the target protein was studied. Previously observed differences in expression levels of N-terminally tagged enhanced green fluorescent protein (EGFP) were not observed herein, when the tags were instead fused to the C-terminus of the protein. In IMAC experiments, C-terminally tagged EGFP’s were however eluted at a slightly lower imidazole concentration compared to their N- terminally tagged counterparts. B. The influence of the tags NT1A and HIT2 on the structure and stability of EGFP was studied by guanidine hydrochloride induced unfolding experiments. NT1A and HIT2 were not found to affect the structure and stability of EGFP, confirming their suitability as affinity tags. Strategy 2 was pursued by studying two fundamental aspects of Ca2+ based IMAC. A. Immobilized Ca2+ chelates were studied by packing ligands immobilized to SepharoseTM 6 Fast Flow into columns and subjecting them to chromatography experiments. The macrocyclic ligands 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and 1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephopshonic acid) (DO3P) were compared with the linear ligands (4-amino-1-hydroxy-1-phosphono-butyl)phosphonic acid (alendronate) xiii and IDA. NaCl was found to displace bound Ca2+ from all ligands investigated. For Ca2+- DO3P, pH values below 7.5 also caused displacement of Ca2+ ions. B. The Ca2+ affinity to putative Ca2+ binding tags was studied. Novel disulfide bridge constrained cyclic tags fused to EGFP were successfully developed and purified, but their expression levels were low. One cyclic tag (previously developed by Imperiali et al.) bound the Ca2+ analogue Tb3+ (log β = 5.2). As the Tb3+ affinity decreased dramatically when the disulfide bridge was broken by a reducing agent, cyclic tags present promising candidates as Ca2+ binding tags. In conclusion, both strategy 1 and 2 generated results of relevance to the overall aim of the thesis.


Campus location


Principal supervisor

Milton T. W. Hearn

Year of Award


Department, School or Centre

Centre for Green Chemistry


Doctor of Philosophy

Degree Type



Faculty of Science