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Site‐specific reversible immobilization and purification of His‐tagged protein on poly(2‐acetamidoacrylic acid) hydrogel beads
Author(s) -
Ha EunJu,
Kim BongSoo,
Park EunKyoung,
Song KiWon,
Lee SunGu,
An Seong Soo A.,
Paik Hyunjong
Publication year - 2013
Publication title -
polymers for advanced technologies
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.61
H-Index - 90
eISSN - 1099-1581
pISSN - 1042-7147
DOI - 10.1002/pat.3052
Subject(s) - self healing hydrogels , green fluorescent protein , materials science , photopolymer , fluorescence , bead , histidine , biophysics , polymer , polymer chemistry , chemistry , biochemistry , amino acid , polymerization , composite material , physics , quantum mechanics , gene , biology
Ni 2+ ‐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 Ni 2+ ‐PAAA hydrogel beads, 51% of yielding recovery. The maximum binding capacity of His‐tagged GFP was estimated to be 0.45 µg/mg of Ni 2+ ‐PAAA hydrogel beads. The recombinant His‐tagged GFP from the soluble fraction of E. coli BL21(DE3) cell lysates was purified with Ni 2+ ‐PAAA hydrogel beads. The major advantage of the Ni 2+ ‐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.