Expression And Characterization Of Porcine Trypsin In Pichia Pastoris

Trypsin (EC3.4.21.4) is a member of serine protease families, and it belongs to alkaline proteolytic enzymes. The main component of the pancreatic juice is inactive trypsinogen. Trypsinogen can be activated by intestinal kinase or self-activation of trypsin, and it can specially cleave the carboxyl side chains of lysine residue and arginine residue. The optimum temperature of most trypsins extracted from animals is37℃, while the optimum pH is8. There are subtle differences between different species.The demand of trypsin gradually increases in recent years, and its proportion of enzyme market is larger and larger. Trypsin has accounted for about3%of annual enzyme demand. So a new method of producing trypsin which has lower cost is urgently needed. And recombinant DNA techniques have provided a new way to obtain high quality and low price of trypsin.This research focused on the expression and characterization of porcine trypsin in P.pastoris. The reason why we investigated porcine trypsin from any of the other mammals was porcine trypsin had being much better studied when compared with other trypsins. And porcine trypsin has a higher activity than most of the trypsins of other sources. It also has more stable physical and chemical properties than other trypsins. Based on those advantages, we attempted to express the porcine trypsin gene in P.pastoris GS115. The main content of the study is described as follows:1. Gene synthesis:The gene sequence of a porcine trypsin was synthesized in vitro, and was cloned to the vector pHBM905BDM;2. Vectors’ construction:After being sequenced, the recombinant plasmid was named as pHBM905BDM-Try.2-4copies of Trypsin vectors were constructed, and named as pHBM905BDM-2Try, pHBM905BDM-3Try and pHBM905BDM-4Try, respectively;3. Transform:1-4copies of Trypsin vectors were transformed into P. Pastoris GS115;4. Clutured with shake flasks:Recombinants of1-4copies of Trypsin were incubated and clutured in shake flasks with1%(v/v) methanol induction;5. Detection of expression:The culture supernatants of shake flasks were analyzed by SDS-PAGE and Western blottingting;6. Purify:Crude Trypsin was purified and activated in vitro;7. Characterization of Trypsin:Measure the enzyme activity, then determine the optimum temperature and pH of porcine trypsin, the optimum temperature is35℃, the optimum pH is8.5.