Grafting polymerization of glycidyl methacrylate onto capillary-channeled polymer (C-CP) fibers as a ligand binding platform: Applications in immobilized metal-ion affinity chromatography (IMAC) protein separations

Abstract

Immobilized metal-ion affinity chromatography (IMAC) is a valuable method for preparative and analytical-scale protein separations. Nylon 6 capillary-channeled polymer (C-CP) fibers were grafted with glycidyl methacrylate (GMA) as a monomer with ceric ammonium nitrate (in dilute nitric acid) used as the initiator. The polymerization reaction occurs rapidly (15 min) in a residential microwave. Iminodiacetic acid (IDA) is then attached to the grafted GMA polymers by reacting with the reactive terminal epoxide groups. Different parameters regarding the grafting time, initiator concentration and conversion time were investigated to find the optimal conditions for the entire modification process. The resulting nylon-IDA fibers were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM). The resulting carboxyl density and copper binding capacity were determined to be 612 ± 21 μmol g−1 and 375 ± 12 μmol g−1, respectively. When charged with Cu2+ ions and packed in a column format, the nylon-IDA fibers can be applied as an IMAC stationary phase for the separation of histidine rich proteins. The performance of this novel phase was evaluated through the separation of a mixture of model proteins (cytochrome C, α-chymotrypsinogen A and lysozyme) and a recombinant histidine-tagged protein (his-tagged ubiquitin). Despite multi-step modifications, columns of the modified fibers still maintain the anticipated high levels of throughput and efficiency, with binding capacities of 6.89 ± 0.56 mg lysozyme g−1 fiber and 6.32 ± 0.12 mg His-tagged ubiquitin g−1 fiber.