Production, propagation and differentiation of hematopoetic cells producing, purification and characterization of epo and tpo as medical biotechnological product

Hematopoietic growth factors are cytokines that stimulate the proliferation and differentiation of blood cells. Erythropoietin (EPO) and thrombopoietin (TPO) are the main hematopoietic growth factors. The effect of erythropoietin on the formation of red blood cells is well known. Thrombopoietin stimulates megakaryopoiesis and platelet formation. During hematopoiesis, EPO and TPO may interact in a synergistic and antagonistic manner. EPO and TPO exhibit significant homology in the receptor binding domains (20% identity and 25% similarity). In this thesis, human EPO protein was produced in bacterial expression system using recombinant DNA technology. The three objectives of this study were to provide the sequence cloned into the E. coli pET30a expression system by bioinformatics studies; Recombinant expression and purification by transformation into E.coli BL21 pLysE cells; Biological characterization for endothelial cells. The selection of transformed cells was carried out on the appropriate antibiotic medium. EPO was expressed with His-tag and Nickel-based affinity chromatography techniques were used for purification. It was analyzed qualitatively by SDS-PAGE method. The biological activity of the recombinant EPO produced in this study was determined by the polylation analysis using human umbilical vein endothelial cells (HUVEC). However; Studies have been carried out for the production of human TPO protein in yeast expression system using recombinant DNA technology. In this study, pPIC3.5K-TPO vector obtained as a result of bioinformatics studies is artificially provided. The pPIC3.5K-TPO construct was linearized. However, the transformation of Pichia pastoris GS115 into competent cells and its integration into the Pichia pastoris genome could not be achieved.