Fermentation, Purification And Pharmacodynamics And Mechanism Of Proliferation Of Platelet - Forming Factor

Aminoacyl-tRNA synthetases catalyze the aminoacylation of their cognate transfer RNAs (tRNA) and play a crucial role in protein synthesis. However, during evolution, human tRNA synthetases have acquired latent signaling activities that can be released by processing of the inactive full length enzyme and secretion of the active signaling molecule. For instance, it has been shown that the human tyrosyl-tRNA synthetase can be split into two fragments with distinct signaling activities. The N-terminal fragment of the tyrosyl-tRNA synthetase acts as a chemoattractant molecule and promotes megakaryocyte development, eventually leading to an increase in platelet production. Tmax is the full-length human tyrosyl-tRNA synthetase engineered with aY341amino acid change that unmasks its thrombopoietic activity.In this study, we expressed codon-optimized rhTyrRS in E. coli under fermentation conditions. The soluble protein was purified by three-step purification process using cation exchange chromatography, gel filtration chromatography and anion exchange chromatography. The protein is characterized by Western blot with monoclonal antibodies、SDS-PAGE、MASS、HPLC and N-terminal sequencing. Biological activity is verified by using an aminoacylation assay.In rat efficacy study, Tmax was shown to have platelet stimulating activity at the dose of1000μg/kg, confirming the thrombopoietic effects of Tmax on anemia of myelosuppression induced by y irradiation in SD rats.One of the crucial steps in platelet production is the migration and adhesion of the immature megakaryocytes to the bone marrow endothelial cells that delineate the sinusoids, an interaction that facilitates differentiation and maturation. Our experiment confirmed that Tmax act as a chemoattractant for the M-07e cells; In addition, Tmax enhance IL-8production from monocyte in HL-60cells.