Purification of recombinant enhanced green fluorescent protein expressed in Escherichia coli with new immobilized metal ion affinity magnetic absorbents

Abstract

A new immobilized metal ion affinity (IMA) adsorbent containing superparamagnetic nanoparticles and coated with hydrophilic resins are proposed here to improve the purification of His-tagged proteins. The magnetic chelating resin was prepared by radical polymerization of magnetite (Fe 3O 4), styrene, divinyl benzene (DVB) and glycidyl methacrylate–iminodiacetic acid (GMA–IDA) in ethanol/water medium. IDA is immobilized on magnetite as a ligand and pre-charged Cu 2+, Zn 2+ and Ni 2+ as metal ions. To identify the GMA–IDA magnetic particles easily, we named these particles MPGI. The MPGI adsorbent was used to test their suitability for the direct recovery of an intracellular, polyhistidine-tagged protein, enhanced green fluorescent protein [EGFP-(His) 6], from Escherichia coli lysates in a single step. Parameters influencing the purification efficiencies such as pH, ionic strength and imidazole concentration were optimized to achieve improved separation. The optimal selectively was observed in binding buffer (0.2 M NaCl, 0.02 M imidazole), washing buffer (0.4 M NaCl, 0.03 M imidazole) and elution buffer (0.50 M imidazole). The Cu 2+-charged MPGI adsorbent had the highest yield and purification factor at 70.4% and 12.3, respectively. The calculated isotherm parameters ( Q m = 53.5 mg/g, K d = 5.84 mg/mL and Q m/ K d = 9.2 mL/g) indicated that the MPGI adsorbent could be used as a suitable adsorbent for EGFP from an aqueous solution.