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 (0.2M NaCl, 0.02M imidazole), washing buffer (0.4M NaCl, 0.03 M imidazole) and elution buffer (0.50M 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 MPGI adsorbent could be used as a suitable adsorbent for EGFP from an aqueous solution.
Chiang
Chen-Li
CL
Department of Chemical and Materials Engineering, Southern Taiwan University, 710 Yung-Kang City, Tainan Hsien, Taiwan. chiang@mail.stut.edu.tw <chiang@mail.stut.edu.tw>
Chen
Chuh-Yean
CY
Chang
Ling-Wei
LW
D000327:Adsorption; D002614:Chelating Agents; D003300:Copper; D004852:Epoxy Compounds; D004926:Escherichia coli / Q000235:genetics*; D005290:Ferric Compounds / Q000737:chemistry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification*; D007098:Imino Acids; D007202:Indicators and Reagents; D008280:Magnetics; D008670:Metals; D008689:Methacrylates; D053758:Nanoparticles; D009532:Nickel; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification*; D020058:Styrene / Q000737:chemistry; D014753:Vinyl Compounds / Q000737:chemistry; D015032:Zinc
D002614:Chelating Agents; D004852:Epoxy Compounds; D005290:Ferric Compounds; D007098:Imino Acids; D007202:Indicators and Reagents; D008670:Metals; D008689:Methacrylates; D011108:Polymers; D011994:Recombinant Proteins; D014753:Vinyl Compounds; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C000499:ferric oxide; D020058:Styrene; D003300:Copper; D009532:Nickel; C004985:divinyl benzene; D015032:Zinc; C007870:glycidyl methacrylate; C008109:iminodiacetic acid