Journal of Chromatography A, 1190 (2008) 18–26 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels Nilay Bereli a ,M¨ uge Andac ¸ a ,G¨ ozde Baydemir a , Ridvan Say b , Igor Yu Galaev c , Adil Denizli a,∗ a Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey b Department of Chemistry, Anadolu University, Eskisehir, Turkey c Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden article info Article history: Received 10 January 2008 Received in revised form 22 February 2008 Accepted 28 February 2008 Available online 12 March 2008 Keywords: Cryogels Molecular imprinting Molecular recognition Lysozyme purification Protein adsorption Affinity binding abstract Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. The aim of this study is to prepare lysozyme-imprinted supermacroporous cryogels which can be used for the purification of lysozyme (Lyz) from egg white. N-Methacryloyl-(l)- histidinemethylester (MAH) was chosen as the metal-coordinating monomer. In the first step, Cu 2+ was complexed with MAH and the lysozyme-imprinted poly(HEMA–MAH) [Lyz-MIP] cryogel were produced by free radical polymerization initiated by N,N,N ′ ,N ′ -tetramethylene diamine (TEMED) in an ice bath. After that, the template (i.e., lysozyme) was removed using 0.05 M phosphate buffer containing 1 M NaCl (pH 8.0). The maximum lysozyme adsorption capacity was 22.9mg/g polymer. The relative selectivity coef- ficients of Lyz-MIP cryogel for lysozyme/bovine serum albumin and lysozyme/cytochrome c were 4.6 and 3.2 times greater than non-imprinted poly(HEMA–MAH) (NIP) cryogel, respectively. Purification of lysozyme from egg white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 94% with recovery about 86%. The Lyz-MIP cryogel could be used many times without decreasing the adsorption capacity significantly. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Molecular imprinting is a technology to create recognition sites in a macromolecular matrix using a molecular template [1]. In other words, both the shape image of the target and alignment of the func- tional moieties to interact with those in the target are memorized in the macromolecular matrix for the recognition or separation of the target during formation of the polymeric materials themselves [2]. Molecular imprinting has been used successfully for imprinting of small molecules and metal ions [3–14]. Due to the small number of publications, quantitative experimental data on protein-imprinted hydrogels is very limited. The difficulties involved in using the technique for the template imprinting of proteins are their large molecular sizes, the fragility and complexity of the molecules. In spite of these difficulties, there is still a strong incentive to prepare bioimprinted polymers and attempts have been made to prepare protein-imprinted polymers via different strategies [15]. Molec- ularly imprinted polymers (MIP) are easy to prepare, stable and capable of molecular recognition [16]. These materials are also less expensive to synthesize and can be manufactured in large quanti- ∗ Corresponding author at: P.K. 1, Samanpazari, 06242 Ankara, Turkey. Tel.: +90 312 2992163; fax: +90 312 2992163. E-mail address: denizli@hacettepe.edu.tr (A. Denizli). ties with good reproducibility. Therefore, MIPs can be considered as artificial affinity media. Molecular recognition-based separation techniques have received much attention in various fields because of their high selectivity for target molecules. Lysozyme (Lyz) is found in a variety of vertebrate cells and secretions, such as spleen, milk, tears and egg white. Lysozyme lyses certain bacteria by hydrolysing the -linkages between the muramic acid and N-acetylglucosamine of the mucopolysaccha- rides which are present in the bacterial cell wall. Its common applications are as a cell disrupting agent for extraction of bacterial intracellular products, as an antibacterial agent in ophthalmologic preparations, as a food additive in milk products and as a drug for treatment of ulcers and infections [17]. The potential for its use as an anticancer drug has been demonstrated by animal and in vitro cell culture experiments [18]. Lysozyme has also been used in can- cer chemotherapy [19]. The studies conducted so far show that it induces the activity of phagocytizing cells, influences immunolog- ical processes by stimulating immunoglobulin synthesis, promotes interferon synthesis and modulates tumour necrosis factor genera- tion [20]. High purity requirements for both natural and therapeutic proteins along with the commercial pressures to reduce process- ing costs have stimulated more efficient, simple and relatively less expensive separation techniques for lysozyme production in the recent years [21]. The large-scale applications require more effi- cient and cost effective techniques for its isolation [22]. 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.02.110