Aptamer Temelli Afinite Kromatografisiyle Protein C Saflaştırılmasının Araştırılması

Abstract

Protein C (PC) is a vitamin K-dependent plasma protein. Activated Protein C (APC) is a glycoprotein derived from protein C. It is formed by cleavage of an activation peptide by the thrombin-linked thrombomodulin (T-TM) complex. Activated Protein C (APC) is a powerful anticoagulant and facilitates the breakdown of blood clots. Activated Protein C (APC) shows anticoagulant properties by inhibiting cofactors (FVa and FVIIIa) in the coagulation cascade. PC is a trace protein with a concentration of 4 μg/mL in blood. Serious problems are observed when the PC level in the blood decreases. Patients with protein C deficiency are at risk of DVT (deep vein thrombosis) and clotting complications that cause a life-threatening decrease in tissue oxygen. If these blood clots get into the bloodstream, they trigger a heart attack, stroke, and pulmonary embolism. The purification of protein C by an effective and economical method is highly important. Cryogels, are gel matrices of monomer or polymer initiators prepared in partially frozen solutions. They consist of interconnected macropores and are used in many biological applications thanks to their osmotic, chemical and mechanical properties. Application areas of cryogenic; As a tissue scaffold, biomolecules, cells, etc. As a carrier in the immobilization of proteins, microorganisms, cells and so on. Used in chromatographic separation processes. In this thesis, Activated Protein C (APC) specific DNA aptamer-based cryogels at different rates were synthesized using the affinity technique and applied for Protein C purification and separation processes. Cryogel membranes with DNA aptamer in three different ratios were prepared. The synthesized APC-specific DNA aptamer cryogels were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), swelling test, surface area, and contact angle analysis. The macroporous structures of the cryogels are given by SEM analysis images. The effects of pH, protein C concentration, temperature, ionic strength on Protein C adsorption and their reusability were investigated. Maximum PC adsorption was found to be 89.02 mg/g with the DNA aptamer attached cryogel membrane at the highest rate. When adsorption-desorption studies were applied to cryogels ten times in succession, the stability in adsorption capacity was calculated as 96.11%. In addition, the selectivity of aptamer-based cryogel membranes against different proteins was investigated. As a result, it was determined that it showed high selectivity against PC. In the last part of the thesis, Protein C purification from an artificial plasma medium was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).