Preparation Of Immobilized Metal Affinity Membrane Based On Dendritic-grafting Modification And Protein Adsorption Efficiencies

Since immobilized metal affinity membrane chromatography (IMAM) allows the processing of a large amount of sample in a relatively short time, low press drop and easier scale up. It has potential applications in the separation and purification of biomolecules. However, the adsorption capacity for membrane chromatography is not comparable to that for conventional bead-packed column chromatography because of its low surface area, which limits its separation efficiency. In order to improve the protein adsorption capacity, this protocol proposed a dendritic-grafting method introducing more functional groups to prepare immobilized metal affinity membrane(IMAM) with high metal ions and protein binding capacities via a series of chemical reactions, exploring the protein adsorption efficiencies and purification of lysozyme from egg white.Firstly, the amine was introduced on the surface of the regenerated cellulose (RC) membranes through two-step reaction. Subsequently, methyl acrylate and ethylene diamine were added step by step to form dendritic structure on the membrane surface. Finally, epichlorohydrin and iminodiacetic acid was reacted to produce metal chelate affinity ligand and Cu2+ were immobilized to prepare immobilized metal affinity membrane based on dendritic-grafting modification. The membrane structure was evaluated by FTIR and XPS. Lysozyme was used as a model protein to explore the static and dynamic adsorption behavior of protein. The maximum adsorption capacity for lysozyme on the prepared IMAM can reach 162 mg/cm3, the highest in the literature. The effect of pH of, ionic strength, hydrophobic interactions and adsorption time on the binding capacity was assessed. Meanwhile, The IMAM can also purify lysozyme from egg white and the protein recovery yield can reach 94.3%,50% higher than immobilized metal affinity membrane without dendritic-grafting modification. Finally, His6-tagged antibacterial peptide B- Human Epidermal Growth Factor (CB-EGF) fusion protein was purified from Escherichia coli crude extract by IMAM and high protein purity was obtained. All of these results indicated that the IMAM may find potential application in His-tagged protein separation and purification.