Abstract
Selective separation and purification of protein from complex medium is required to completely investigate the structure and function of the target protein. In this study, a composite macroporous agarose monolith containing iminodiacetate-chelated Ni2+ ligands was synthesized for selective separation and purification of histidine-tagged recombinant proteins. The large and interconnected pores in the monolith enabled fast binding of proteins with high matrix tolerance in treating complex mediums. To realize the selective protein binding, the iminodiacetate was directly conjugated to epoxy-functionalized agarose monolith via simple chemical reactions between epoxy and imino groups. After chelated Ni2+, the composite monolith could bind histidine-tagged recombinant proteins through the coordination interaction between transition metal ions and the imidazole ring of histidine. To further increase the binding capacities of the monolith, a hydrophilic intermediate polymer chain containing multiple iminodiacetate immobilization sites was conjugated to the azide-functionalized agarose monolith via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The morphology and chemical composition of the composite agarose monolith were characterized systematically. The protein binding capacities of the obtained composite agarose monolith were subsequently investigated. The binding capacities of the composite agarose monolith towards the model proteins Gp10 and Lys84 were 0.93 and 0.51 mg/mL, respectively. The protein binding of the composite agarose monolith could be manipulated by adjusting the temperature and concentrations of imidazole. These results demonstrate that the composite agarose monolith could be used as an affinity medium for rapid separation and purification of histidine-tagged recombinant proteins from biological samples.
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