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

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 (Fe3O4), 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 Cu2+, Zn2+, and Ni2+, 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 Cu2+ -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 MPGl adsorbent could be used as a suitable adsorbent for EGFP from an aqueous solution. (C) 2008 Elsevier B.V. All rights reserved.