Concomitant selective adsorption and covalent immobilization of a His-tagged protein switch with silica-based metal chelate-epoxy bifunctional adsorbents

Abstract

A series of silica-based bifunctional adsorbents containing both metal-chelating groups and epoxy groups for the concomitant purification and immobilization of His-tagged protein switch RG13, a potential bioreceptor for developing maltose biosensors, were prepared by controlling the ratio of iminodiacetic acid-conjugated silane (GLYMO-IDA) and silane (GLYMO) used for surface modification. The bifunctional adsorbent prepared with a [GLYMO-IDA]/[GLYMO] ratio of 0.2, containing a [metal chelating group]/[epoxy group] ratio of 1.42, was shown to exhibit a metal chelating capacity of 88.42 ± 15.91 μmole Cu2+/g, a protein adsorption capacity of 1.81 ± 0.19 mg/g and a superior selectivity over the other bifunctional adsorbents. Results of kinetic studies showed that selective adsorption and covalent bond formation at 4 °C were achieved in 1 h and 15 h, respectively, which allowed the sequential adsorption and covalent immobilization of protein switch RG13. A protein immobilization yield of 94.6 % and a global activity yield of 63.4 % were obtained, giving an immobilized protein switch RG13 with an enzymatic activity of 4.57 ± 0.19 U/g, under optimal conditions at pH 8.0 and 40 °C. In the repeated-batch operation, the bifunctional adsorbent-immobilized RG13 retained 91 % of the original activity after 20 cycles, 39 % higher than the counterpart prepared with monofunctional metal chelate adsorbent mediated solely by coordinate linkages.