Research On Recombinant Human Cationic Trypsin

Trypsin (EC3.4.21.4) is the first enzyme to be discovered and named of serine proteases family, trypsinogen is secreted by the pancreas in animals. Trypsin as an endoproteinase strongly prefers to cleave amino substrates following Arg or Lys residues. There are three types of trypsinogen are secreted by human pancreas, and the human cationic trypsinogen and anionic trypsinogen were widely studied before. Here, the human cationic trypsinogen (human cationic trypsinogen, HTg1) and its mutant (mHTg1) were successfully expressed in the E.coli BL21(DE3). Through the process of refolding and activation, the active recombinant human cationic trypsin (rHT1) and its mutant (mHT1) were obtained. Some characteristics of purified rHT1had been studied in this paper.Recombinant human cationic trypsinogen was overexpressed (amount to15-20%) as inclusion body in the E.coli BL21(DE3). By high cell density fermentation, the wet weight reached to72g/L.After purification by CM-FF cation exchange chromatography, the purity of recombinant human cationic trypsin was over95%by SDS-PAGE analysis. The optimum range of temperature of rHT1is40℃to45℃, and the pH optimum range is8.0-9.5;The rHT1is very stable in the range of pH2to pH5, lose its activity under pH12. what’s more, rHT1is stable under the temperature from0℃to37℃, kept at37℃for2h, its activity remained more than95%, but at70℃, the activity of rHT1lost fast. Some metal ions, like Cu2+、Ni2+、Co2+、Zn2+inhibit obviously the activity of rHT1in the alkaline environment, while the stability of rHT1was enhanced in the presence of Ca2+; Michaelis constant Km of rHTl was0.013mmol/L with n-benzoyl-1-arginine ethyl ester(BAEE) as a substrate, the maximum UV absorption wavelength of rHT1is278nm. The rHTl was applied in the digestion of Hela cells, and the effect is perfect.By comparing the wild rHT1and its mutant, it was showed that a disulfide bond Cys139-Cys206played an important role on the stability of rHT1, it is suggested recombinant human anionic trypsin (rHT2) is less stable than rHT1due to lake of this disulfide bond in rHT2. What’s more, WISS-MODEL homology modeling was to investigate the relationship between the Cys139-Cys206and stability of rHT1.