Interaction of histidine-tagged human proinsulin with immobilized nickel ion: Effect of chelating ligand and thermodynamics analysis

Abstract

The nature of the chelating ligand is one of various factors affecting the performance of IMAC. In this work we studied the effect of three chelating ligands on the adsorption of recombinant human proinsulin with His-tag (proinsulin-(His) 6). The chelating ligands (complexed with Ni(II)) were two tetradentates (carboxymethylated aspartic acid, CM-Asp, and tris(2-aminoethyl)amine, TREN) and one pentadentate (tris (carboxymethyl) ethylenediamine, TED). Their performance were compared with the performance of one tridentate ligand (iminodiacetic acid, IDA) and the commercial adsorbent HisTrap both also complexed with Ni(II). Higher selectivities were achieved with Ni(II)-CM-Asp and Ni(II)-TED. The adsorption capacity decreased according to the order: TREN, HisTrap, IDA, CM-Asp, and TED (172.82, 167.98, 133.09, 110.18, and 69.22 mg of proinsulin/mL of gel, respectively, at 25 °C). Although TREN-agarose had the highest capacity, the ligand with the second highest capacity – HisTrap – showed better performance since proinsulin was eluted in a single, concentrated peak with imidazole, while for Ni(II)-TREN proinsulin was eluted with imidazole and EDTA, requiring an extra step to free the product from the EDTA. Therefore, the choice of an adsorbent for this separation depends on the priority: capacity (TREN or HisTrap for one step process) or selectivity (CM-Asp). Adsorption isotherms were determined for temperatures from 4 to 45 °C and the Langmuir model was fitted to the experimental data. The thermodynamics analysis showed positive Δ H° (from 18.83 to 86.05 kJ/mol), indicating that the adsorption of proinsuin-(His) 6 in all chelating ligands is an endothermic process. The negative change in Gibbs free energy (from −26.30 to −35.11 kJ/mol) indicated that the adsorption of the proinsulin-(His) 6 on all the adsorbents studied was a thermodynamically favorable process.