Protein İzolasyonu için İmmobilize Metal Afinite Kromatografisi Bazlı Kesikli Sistemlerin Geliştirilmesi
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Proteins are macromolecules that have functions as hormon, enzyme, structure and defense. Today, the isolation and purification of proteins, determination of their functions and explanation of their biochemical reactions are developing issues. For the identification of biological processes in organisms, a new approach is getting important called ‘immobilized metal affinity chromatography (IMAC)’. Purification of recombinant proteins, isolation of serum proteins and phosphopeptides, characterization of proteins and peptides, isolations of macromolecules like RNA, DNA, oligonucleotide are major applications of IMAC. In the scope of this study, the magnetic silica microspheres were coated with a polydopamine (PDA) layer and Ni2+ ions were immobilized on the PDA coating. In the first step of the synthesis, the product of the dispersion polymerization process, poly(glycidyl methacrylate), was used as seed latex in the multistage microsuspension polymerization. Then, the monodisperse-porous poly(methacrylic acid-co-ethylene dimethacrylate) microspheres were obtained. The magnetization of microspheres was performed by the impregnation of Fe2+ ve Fe3+ ions into the particles. Next, monodisperse-porous magnetic silica microspheres were synthesized using the sol-gel templating protocol, with 6 µm in size and the average pore size of 8.6 nm. The magnetic SiO2 microspheres were coated with a thin layer of PDA for functionalization. The magnetic SiO2 microshperes and PDA coated microspheres had the saturation magnetization values of 25 and 21 emu/g, respectively, those were higher than the sufficient value for magnetic seperation (16.3 emu/g). Finally, Ni2+ ions were immobilized via the functional hydroxyl groups of PDA layer on the magnetic microspheres and the final product was used as the chromatographic sorbent in IMAC process. The chromatographic sorbent was used in the successive adsorption-desorption in batch fashion. The adsorption capacity, and the desorption and isolation efficiencies were determined to demonstrate the affinity of sorbent against to bovine serum albumin (BSA), hemoglobin (HB) and lysozyme. The results indicated that the adsorption capacity and the desorpsiton efficiency decreased with the increasing sorbent concentration. The adsorption capacity took its maximum value for all proteins at a protein concentration of 2.0 mg/mL. The corresponding values were 80, 70 and 54 mg/g for HB, lysozyme and BSA, respectively. The maximum isolation efficiencies were obtained at 0.5 and 1.0 mg/mL protein concentrations. All analyses were repeated with PDA coated microspheres not containing Ni2+ ions to figure out the function of Ni2+ ions during the protein isolation. The results showed that the sorbent not containing Ni2+ ions had lower adsorption capacity when compared to Ni2+ immobilized one. The desorption and isolation efficiencies were also lower with the sorbent not containing Ni2+ ions. The adsorption isotherms were plotted for all proteins. As a result, the adsorption of proteins onto the synthesized sorbent was adequately described by Langmuir isotherm. This model demonstrated that the adsorption was homogeneous and occurred in the form of a monolayer on the sorbent surface.