Recombinant Human Pancreatic Kallikrein Expressed In Yeast Pichia Pastoris

Kallikreins are a group of serine proteases that are found in diverse tissues and biological fluids in human body. Pancreatic kallikrein has the ability to release efficiently a bioactive kinin from a kininogen. The kallikrein-kinin system is involved in the control of blood pressure, electrolyte balance, inflammation, and other diverse physiological processes. Thus, the pancreatic kallikrein has widely pharmaceutical action, including degrading the blood pressure and treating the diabetic nephropathy,cardiovascular ischemic and cerebrovascular complications. In the study, the human pancreatic kallikrein cDNA was synthesized and inserted into the plasmid pPIC9K to construct recombinant plasmid pPIC9K/hPK followed by linearizing by restriction endonuclease BglII. Then it was transformed into the host Pichia pastoris GS115 by eletroporation. On MM and MD plates, 27 His+Muts positive colonies had been screened. 3 colonies with high-copy purpose gene were gained by the G418 (geneticin) resistance screening. Among them, one colony was cultured in BMGY/BMMY flask for the expression of recombinant human pancreatic kallikrein in vitro. The main band of the fermentation supernatant on SDS-PAGE was about 37 000 and the expression level of purpose protein was about 30% of total soluble proteins by gel analysis. The expressed purpose protein was glycosylated confirmed by the glycosylation analysis.To further industrialized production, the base salt medium was used for expressing the recombinant hPK. Some cultivating condition such as pH, salt concentration and organic nitrogen source, was optimized. The dialysis deslating and ion exchange chromatography were performed as the primary purification methods. Comparing the adsorption-elution effect of several columns, the selectivity of weak anion exchange column, DEAE Sepharose FF, was better for the object protein hPK purification.The purified hPK enzyme activity was mensurated after prohPK was activated by trypsin. The hPK specific activity reached 5.6U/mg. By analysing of the kinetic parameter of recombinant hPK, the Michaelis contant is 5.4×10-2mM,near to the value reported by literature, which further confirmed that the recombinant hPK was exactly expressed in Pichia pastoris.