Metal chelate-epoxy bifunctional membranes for selective adsorption and covalent immobilization of a His-tagged...

The preparation and application of metal chelate-epoxy bifunctional membranes for the selective adsorption and covalent immobilization of His-tagged protein switch RG13 were shown in this study. By controlling the concentration of iminodiacetic acid (IDA) and reaction time during the conjugation of IDA on to the epichlorohydrin-activated regenerated cellulose membrane, 5 metal chelate-epoxy bifunctional membranes, with degrees of IDA conjugation in the range of 20%-81%, were prepared. The bifunctional membrane with an IDA conjugation degree of 30%, designated as BFM₃₀, exhibited a sound adsorption capacity of 0.203 mg/cm² with a relatively high content of epoxy groups for covalent immobilization, were selected. The concomitant selective adsorption and covalent immobilization of the His-tagged RG13 with BFM₃₀ were carried out by 2-h incubation for protein adsorption and subsequent 16-h incubation for covalent immobilization after the removal of undesired proteins with wash buffer, giving an immobilization yield of 63% and a global activity yield 40%. The RG13 immobilized on the metal chelate-epoxy bifunctional membrane exhibited superior operational stability in a repeated batch process, retaining 94% of its initial activity after 20 cycles. The employment of the bifunctional membranes could significant facilitate enzyme immobilization processes by eliminating the need for prior protein purification.