Trombin Saflaştırılması İçin Moleküler Baskılanmış Mikrokriyojeller

Abstract

The field of protein purification appears as a very necessary method to understand the structural and functional properties of proteins and to explain their roles in cells and tissues. Nowadays, with the developments in life sciences, protein purification plays an important role in certain applications such as protein production, which is a basic step in the biotechnology industry. Therefore, the need for low-cost, easy and efficient purification methods in the field of protein purification is increasing.

In the presented thesis study, suitable polymeric materials were prepared to obtain high purity, single-step and low-cost thrombin, which has an important place in the field of health. For this purpose, the molecular imprinting method, which is highly selective and allows reuse and is preferred in many areas due to its superior properties, was used in the study. Additionally, micro-scale microcryogels with large pore sizes and unique properties such as flexibility, short diffusion path, good biocompatibility and high mechanical strength have been synthesized. Within the scope of the thesis study, the effectiveness of these prepared polymeric materials in the bioseparation and purification processes of the protein in question, thrombin, was investigated.

In the first step of the thesis, microstencil array chips with different sizes in the range of 400-650 µm were created with laser application, from which microcryogels would be synthesized. N-Methacryloyl-(L)-glutamic acid, an amino acid-based functional monomer, was synthesized. In the next steps, thrombin imprinted and non-imprinted microcryogels with different contents were synthesized using microstencil array chips. Characterization studies of microcryogels were accomplished with micro-computed tomography (μCT), scanning electron microscope (SEM), optical microscope, surface area measurements (BET analyses) and swelling test measurements. Adsorption, desorption and reusability studies were carried out by scanning various parameters affecting thrombin adsorption such as pH, temperature, initial concentration, ionic strength, adsorption time, and the selectivity of microcryogels was also examined with the determined competitive agents. The purity of thrombin was evaluated by fast protein liquid chromatography (FPLC).