Cloning And Expresion Of Porcine Pepsinogen A In Pichia Pastoris And Detection Of The Recombination Enzyme Activity

Pepsin is wildly presented in gastric juice of vertebrates and can hydrolysis various proteins in the acidic environment. Currently, commercially used pepsin is generally obtained from animal stomach tissue, which is very expensive and un-pure because of the animal organs resource constraints and other proteases. Therefore, a great interest has been focused on the new method of producing pep A (Pepsinogen A).Among these, the microbial production is supposed to be the most promising method because of its high yield, simple production process and lower cost. Therefore, the objective of this study is trying to produce efficiently recombinant porcine pepsin with genetic engineering technology in microbial system.In this study, the cDNA sequences of the pep A gene was first obtained from pig stomach cell by RT-PCR and cloned. The cDAN sequence was then ligated into plasmid pPICZ a A to produceb expression vector pPICZaA-pep A. The recombinant pPICZaA-pep A was linearized with Pme I enzyme and delivered into Pichia pastoris X-33 through electroporation. The transformants were screened in YPDS including zeocin plates. The selected transformants were then screened by RT-PCR for high mRNA level after methanol induction in BMMY culture medium. The result showed that the pep A gene was expressed in Pichia pastoris. The recombinant pep A protein was purified through Ni Sepharose High Performance affinity chromatography. As determined by SDS-PAGE analysis, the recombinant pep A protein had a molecule weight of approximately 45 kDa, consistent with the expected molecular size. Enzyme activity assay revealed that the recombinant pepsin showed a similar degradation ability to the natural.