A Pluronic-coupled metal-chelating ligand for membrane affinity chromatography

A technique for bio-specific affinity chromatography using synthetic nonporous membranes and a new metal-chelating Pluronic surfactant is described. Synthetic polymeric poly(vinyldiene fluoride) membranes were fabricated for use as solid, hydrophobic adsorption membrane matrices. An ethylene diamine tetraacetic acid dianhydride was coupled to the terminal hydroxyl end groups of Pluronic® F108 via a two-step reaction at 40 °C, to create a new metal affinity ligand, Pluronic-N,N-dicarboxymethyl-3,6-diazaoctanedioate (Pluronic-DMDDO). The hydrophobic poly(propylene oxide) moiety of Pluronic allowed non-covalent adsorption of the ligand to the hydrophobic membrane matrix. The protein repellent properties of the hydrophilic poly(ethylene oxide) brush layer of Pluronic, served to preserve the bio-specific activity of the ligand and to increase ligand accessibility. Proton-induced X-ray emission (PIXE) analysis was used to determine the metal binding capacity, stability and surface homogeneity of this immobilised metal affinity membrane system and to generate surface homogeneity maps of the chelated metal ions. The chelate capacity of Pluronic-DMDDO was determined under non-competitive conditions and was of the order (Zn2+ > Ni2+ > Cu2+). An amino terminal hex-histidine-tagged recombinant Escherichia coli pantothenate kinase was used as a test protein. Histidine-tagged pantothenate kinase bound strongly to Pluronic-DMDDO-treated membranes specifically in the presence of Ni2+. Eluted immobilised histidine-tagged proteins retained their biochemical activity and the membranes were capable of being regenerated and re-used.