Abstract
Protein A is the most commonly used ligand in IgG purification due to its specific binding to the Fc receptor of most immunoglobulins, making it commercially important. Molecular imprinting is a method based on the selective recognition of various molecules. Molecular imprinted polymers are materials that are easy to prepare, durable, cheap and have molecular recognition capability. Cryogels are prepared by radical polymerization in a partially frozen environment. The unique structure of cryogels combined with osmotic, chemical and mechanical stability make them attractive chromatography matrices for a variety of biological compounds/specimens (plasmids, pathogens, cells). In this protocol, protein A imprinted supermacroporous poly(2-hydroxyethyl methacrylate) cryogels were prepared in spherical form for protein A purification. The characterization of the prepared cryogels were made by swelling test, scanning electron microscopy (SEM), Fourier transform infrared spectrophotometer (FTIR), and Brunauer–Emmett–Teller (BET) surface area analysis. After characterization, optimum conditions for protein A adsorption were determined in the batch system. The maximum protein A adsorption capacity was determined after optimization of the imprinted cryogels. Protein A relative selectivity coefficients of imprinted cryogels were examined for both Fc and protein G. Protein A was isolated from the bacterial cell wall using fast performance liquid chromatography (FPLC). The separated protein A was determined by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). In the last stage, the reusability of the cryogel was examined.
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Aslıyüce, S., Mattiasson, B., Denizli, A. (2022). Preparation of Staphylococcal Protein A Imprinted Supermacroporous Cryogel Beads. In: Ayyar, B.V., Arora, S. (eds) Affinity Chromatography. Methods in Molecular Biology, vol 2466. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2176-9_18
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DOI: https://doi.org/10.1007/978-1-0716-2176-9_18
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