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Abstract
The work in this thesis was focused on the preparation of immobilized metal ion affinity membranes (IMAMs) to be used in the separation and purification of histidine tag (His-Tag) protein from protein mixture.
The work in this thesis is directed towards the development of polymeric affinity separation membranes, modified by grafting technique, which can be employed in arrange of potential applications of separating of protein aqueous mixtures. Commercially available cellophane has been chosen as membrane material, methyl methacrylate (MMA), and methacrylic acids (MAA) which are expected to have a good interaction with regenerated cellulose (Cellophane) are utilized.
The functionalization of cellophane membrane by introducing metal ion as selective ion for target proteins (His-Tag proteins) is suggested to be accomplished by the three following steps:
1-grafting with methacrylic acid and methyl methacrylate
In the membrane preparation, the functionalization process through grafting of cellophane membranes with MMA&MAA using potassium persulphate and potassium persulphate with sodium bisulphite initiation system respectively have been studied by exploring the effect of different factors, such as monomer concentration, initiator concentration, time of grafting and temperature of reaction on the grafting process. Three parameter namely; grafting percentage ”GP”, grafting efficiency “GE” and weight conversion percent (WC) have been monitored.
2- immobilization of cupper ions
The factor affecting the immobilization of Cu+2 ions on cellophane functionalized membrane have been studied (like pH, Time,...etc.)
3-his-tag protein purification
Membranes with the highest amount of immobilized Cu+2 ions were used in studying the process of proteins separation. Different factors affecting on both the proteins adsorption and elution steps have been studied by the membranes interaction with both proteins.