Site-specific reversible immobilization and purification of His-tagged protein on poly(2-acetamidoacrylic acid) hydrogel beads

Ni2+‐complexed poly(2‐acetamidoacrylic acid) (PAAA) hydrogel beads were developed for the site‐specific reversible immobilization and purification of the histidine‐tagged green fluorescent protein (His‐tagged GFP). PAAA hydrogel beads were prepared by photopolymerization, and significantly improved mechanical properties of PAAA hydrogel beads were observed in comparison with PAAA hydrogel from our previous study. Confocal laser scanning microscopy was used to determine the binding of His‐tagged GFP to the hydrogel beads in three‐dimensional space. Photoluminescence spectroscopy revealed 89% of binding efficiency of His‐tagged GFP to the Ni2+‐PAAA hydrogel beads, 51% of yielding recovery. The maximum binding capacity of His‐tagged GFP was estimated to be 0.45 µg/mg of Ni2+‐PAAA hydrogel beads. The recombinant His‐tagged GFP from the soluble fraction of E. coli BL21(DE3) cell lysates was purified with Ni2+‐PAAA hydrogel beads. The major advantage of the Ni2+‐PAAA hydrogel beads system was simple preparation procedures of producing the matrix, because PAAA hydrogel beads had relatively enhanced mechanical strength than soft hydrogels. Copyright © 2012 John Wiley & Sons, Ltd.