| Tissue kallikrein is a kind of serine proteinase, and its important biological functions indicate that it might be an effective drug against renal disease. In addition, the relationship between the tissue kallikrein content in blood and ischamia stroke makes it as a possible biomarker for the early diagnosis of such a disease.In this dissertation, baculovirus-insect cell system was employed to express human tissue prokallikrein. Sf-9 cell was used to amply recombinant baculovirus, whilst Hi-5 cell was taken to express the target protein. These two cells were recultured in medium without FBS, and further suspended in a spinner flask. After systematic studies on the effects of MOI, infection and the harvesting time, the expression level of recombinant human prokallikrein was as high as 25mg/L. In addition, CHO cell which harboring human tissue kallikrein cDNA was also recultured and suspended in the medium free of FBS.Tissue prokallikrein excreted from insect cells was purified by liquid chromatography. The influences of pH, buffer composition, ionic strength and separation matrix on purification were studied to obtain the optimal conditions for the purification of recombinant prokallikrein. Furthermore, HPLC/MS analysis and amino acid sequencing were performed to validate the purified protein. By HPLC, SDS-PAGE and CIEF analysis, the obtained protein was found pure, and the recovery was about 57%.
|
PDF Full Text Request