Cloning And Expression Of Human Pepsinogen A In Escherichia Coli And Saccharomyces Cerevisiae

The pepsin is the main protein digestive enzyme, and its precursor is theinactive pepsinogen that is secreted by gastric chief cells. The pepsinogen can beconverted into the active pepsin by the hydrochloric acid in the stomach. Pepsinbelongs to the family of aspartic acid proteinase, which can hydrolyze large molecularweight proteins into the small polypeptide. It acts between pH1.0and5.0. Pepsin hasmany applications in the livestock, tanning and wine making industry and so on.Besides, pepsinogen is the biology marker for the diagnosis of gastric cancer. Thediagnosis method has high sensitivity and specificity. The detection of pepsinogen inthe blood is used to screen early stage-gastric cancer, and is with diagnostic value foratrophic gastritis and gastrelcoma.At present, the pepsinogen is usually extracted from the animal stomaches bythe biochemistry technique. The technology is limited by raw materials from animalsand has many disadvantages, such as complicated procedure and low production. Sothe pepsinogen aquired by the technique can not meet the market requirement.With the development of molecular biology, more and more proteins can beexpressed in bacteria, yeast and mammalian cells. The aim of the study is to expresshuman pepsinogen with biological activity in E.coli or S. cerevisiae. The expressedpepsinogen can be converted into pepsin by hydrochloric acid. The PGA gene wasamplified from human stomach tissue by RT-PCR, and ligated into the PET-28aexpression vector. Restriction-enzyme-digestion-analysis and sequencing showed thatthe recombinant plasmid pET-28a-PGA was successfully constructed. ThepET-28a-PGA was transformed into the competent BL21(DE3) cells and expessedunder the induction of IPTG. SDS-PAGE showed that the43KD protein, therecombinant PGA, was expressed in the E.coli. The expressed PGA was in theinclusion body in the E.coli. The expressed PGA was purified by Ni affinitychromatography and gel filtration chromatography. Milk clotting assay demonstrated that the purified PGA was inactive after being cleavaged into pepsin.To obtain the active pepsinogen, we tried to express the PGA in the S. cerevisiae.The PGA gene was subcloned into the pYES2expression vector to construct therecombinant plasmid pYES2-PGA. The plasmid was transformed into the competentS. cerevisiae cells. The transformants were selected on the SC-U plate and the positivetransformants was confirmed by PCR. The expression of PGA in the yeast cells wasinduced by2%galactose for72h. SDS-PAGE indicated that PGA was expressed inthe S. cerevisiae cells with a low level represented as a weak band.To verify the expressed PGA, we conducted Western blottting with both of PGAspecific anti-serum and anti-His-tag antibody. The PGA specific anti-serum was raisedby immunizing chickens with by purified PGA expressed in E.coli. The expressedPGA in the yeast was recognized by PGA specific anti-serum and also anti-His-tagantibody. Milk clotting assay indicated that the lysate of the yeast, after being treatedwith hydrochloric acid, showed activity of pepsin.Together, we expressed human pepsinogen in both of the E.coli and yeast. Therecombinant PGA expressed in the yeast is biologically active.