| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 91 === Thrombomodulin (TM), a glycoprotein found on the endothelial cell surface, plays an important role in regulation of blood coagulation. It was also demonstrated that TM was widely distributed in placental syncytiotrophoblasts, monocytes, neutrophils, epithelial cells, smooth muscle cells, keratinocytes, synovial lining cells, meningeal cells, and tumor cells. During the mouse development, TM is expressed in extraembryonic placental tissues, in the developing cardiovascular system, airway epithelia, cartilage, and in restricted area of the brain. This raises the possibility that TM possesses functions distinct from those related to hemostatic regulation.
In order to study the functions of extracellular domains of TM, we constructed plasmid containing DNA of TM domains including：TMD123, TMD23, TMD12, TMD2, TMEGF1-3, TMEGF1-4, and TMEGF1-5.The DNA constructs were subcloned into pPICZA expression vector containing c-myc fusion peptide and enterokinase cutting site (AspAspAspAspLys). These constructs were transfected into X-33 and the highly expressed clones were selected by Zeocin. All the recombinant proteins were successfully expressed and secreted into the medium in Pichia expression system.
TMD123 protein was expressed in large-scale bioreactor. TMD123 protein was very unstable and tends to decompose during purification. Mg2+ could slow down the TMD123 decomposition. EDTA and Ca2+ had no effect to improve the stability of TMD123. Why TMD123 was unstable in fermentor medium and the protective effect of Mg2+ still needs further investigation.
TMD23 protein was large-scale expressed by bioreactor system and purified by DEAE -Sepharose and Ni2+-chelating Sepharose and the recombinant TMD23 was analyzed by SDS/PAGE and Western blotting. Purified TMD23 showed a sharp band on SDS/PAGE. The purified TMD23 was labeled by FITC for study the binding and internalization with the BAECs, bPAECs, HaCaT cells ,or A549 cells for 4 h at 37℃. The FITC- labeled TMD23 could be internalized into the BAECs faster than other cells. Using confocal microscope, we found that the FITC -labeled TMD23 interacted with the cell membrane; the protein aggregated and engulfed into the BAECs within 30 minutes.
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