Molecular Cloning And Eukaryotic Expression Of The Gene Encoding Porcine Pancreatic Trypsinogen

Trypsin is a serine protease that plays a key role in the activation cascade of pancreatic enzymes involved in digestion. The enzyme can be used to improve feed protein digestion by young animals. To study the effect of porcine pancreatic trypsinogen (PPT) on feed digestion in animals and to produce the recombinant enzyme efficiently at low cost, the full-length cDNA of porcine trypsinogen gene was cloned from porcine pancreas by RT-PCR. The gene seuqence was submitted to GeneBank (FJ969506.1). Sequence analysis showed that the cDNA of PPT shares homologies ranging from 67.5 to 76.6% with Human, Bovine, Mus mouse, Chicken, Oryzias, Engraulis and Pangasius. The obtained cDNA was inserted into eukaryotic expression vector pPICZaA (Invitrogen, Shanghai, China). The gene construct was delivered into Pichia pastoris X-33 cells. Transformants with high-level expression were-selected by SYBR-green quantitative real-time RT-PCR analysis (ABI 7900HT, Applied Biosystems). After 96-h of 0.5% methanol induction, the extracellular recombinant trypsinogen contained a histidine tag in the C terminus was purified using Ni-Sepharose affinity chromatography (GE Healthcare, Piscataway, NJ). The purified protein exhibited a molecular mass of approximately 30 kDa as determined by SDS-PAGE analysis. The successful expression of the recombinant typsinogen in P. pastoris enabled us to further study the enzyme function in animal feeding.