Site-specific reversible immobilization and purification of His-tagged protein on poly(2-acetamidoacrylic acid) hydrogel beads Eun-Ju Ha a , Bong-Soo Kim a , Eun-Kyoung Park b , Ki-Won Song b , Sun-Gu Lee c , Seong Soo A. An d and Hyun-jong Paik a * 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 micros- copy was used to determine the binding of His-tagged GFP to the hydrogel beads in three-dimensional space. Photo- luminescence 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 mg/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. Keywords: hydrogel; purification; immobilization; photopolymerization; histidine-tagged protein INTRODUCTION Significant advances were made in the development and appli- cations of site-specific protein immobilization in recent years. [1–4] The protein immobilization onto fixed or rigid supports would be important for the proteomic and protein-based assays for obtaining information in protein functions and their multiple interactions. [4–9] In addition, protein immobilization would sup- port other applications, including drug screening, diagnostics, bio- sensing, and biocatalysts. [10–15] Both physical adsorption and chemical coupling methods were utilized for the protein immobi- lization. The nonspecific reactions, such as the attachment of pro- tein with solid supports, would be referred as the physical adsorp- tion. In contrast, several site-specific immobilization methods through affinity peptide tags would offer advantages by effec- tively retaining biological activity by maintaining the conforma- tion of the adsorbed protein molecules. Various affinity peptide tag/ligand, such as biotin-avidin, [1,16–18] anti-GST antibody, [9] or His 6 -Ni [6,19–21] were utilized for the site-specific immobilization and for the purification of target proteins. Many studies reported the applicability of affinity tags and target proteins for the pur- pose of site-specific and oriented protein immobilizations. The polyhistidine tag (his-tag) was a popular choice for the immobili- zation and purification of proteins with metal affinity resins. [4,6,13,19–23] Recently, the hydrogel was readily used as supporting materi- als for the immobilization and purification of protein or en- zyme. [24–27] Hydrogels were cross-linked polymers, which could remain in an expanded matrix formation without dissolving in water. The water-absorbing capacity of hydrogels led to their applications in various technological areas, such as materials for contact lenses, matrices for cell encapsulation, and devices for the controlled release of drugs or proteins. Previously, we intro- duced the Ni 2+ -poly(2-acetamidoacrylic acid) (PAAA) hydrogel system, as a novel solid support for the simultaneous immobiliza- tion and purification of proteins or enzymes, based on the site- specific binding interactions between histidine residues and metal ion. [28] The advantage of the Ni 2+ -PAAA hydrogel system over the conventional system was the efficient utilization of the three-dimensional structure of the polymer chains for protein im- mobilization and purification, which reduced the leaking of pro- tein into solution from solid support in fluidic environment. The water-swollen network structure of Ni 2+ -PAAA hydrogel could also prevent the protein denaturation. One drawback of Ni 2+ - PAAA hydrogel was relatively poor mechanical strength, which limited its reusability. In order to improve the poor mechanical * Correspondence to: Hyun-jong Paik, Department of Polymer Science and Engi- neering, Pusan National University, Busan 609-735, Korea E-mail: hpaik@pusan.ac.kr a E.-J. Ha, B.-S. Kim, H.-j. Paik Department of Polymer Science and Engineering, Pusan National University, Busan 609-735, Korea b E.-K. Park, K.-W. Song Department of Organic Material Science and Engineering, Pusan National Uni- versity, Busan 609-735, Korea c S.-G. Lee Department of Chemical Engineering, Pusan National University, Busan 609- 735, Korea d S. S. A. An Department of BioNano Technology, Kyungwon University, Sungnam 461-701, Korea Research Article Received: 14 September 2011, Revised: 21 March 2012, Accepted: 14 May 2012, Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/pat.3052 Polym. Adv. Technol. (2012) Copyright © 2012 John Wiley & Sons, Ltd.