| PSA is sereted into prostatic ducts as an inactive 244-amino acid proenzyme (proPSA) that is activated by cleavage of seven N-terminal amino acids. It is a member of the tissue kallikrein family. The PSA concentration in seminal fluid is0.5-2mg/ml. PSA is a major protein in semen, where its function is to digest the semenogelins and fibronectin in the seminal plasma, thus liquefying the seminal clot after ejaculation. PSA can release a kinin-like substance that smooth muscle contraction by digesting a glycoprotein present in seminal vesicle fluid. The scholars focused on clinic use of PSA including PSA as biomarker for diagnosis, monitoring, prognosis in prostate cancer , since Chu and comrades found that PSA is related with prostate carcinoma. Moreover we found the level and activity of PSA in seminal plasma of is related with infertility (male sterile). The present scientific literature on PSA physiology and pathobiology is confusing. A group of reports have suggested that PSA may act as a tumor suppressor, a negative regulator of cell growth, and a apoptotic molecule, whereas others suggest that PSA may promote tumor progression and metastasis through its chymotrypsin-like activity. We report the production of active, mature rPSA in Pichia pastoris.In our experiments, mRNA was isolated with TRIZOL Reagent and the cDNA was gathered by reverse transcription. The cDNA encoding the full length human PSA was obtained .The PCR product was inserted in TA vector, the resultant plasmid pGEM-PSA was constructed, then the sequence of PCR product was determined. A 0.730-kb fragment was amplified by PCR using the primer and pGEM-PSA. This fragment was ligated into TA vector, the resultant plasmid pGEM-mPSA was constructed. The pGEM-mPSA and pPICZα-C were disgested by Xho I and Xba I, resulted in an inframe fusion of the mature PSA sequence with the Sacccharomyces cerevisiaeα-factor signal sequence. The resultant plasmid-- pPICZα-mPSA was linearized in the 5'AOX1 region with the restriction enzyme PmeI for integration into the 5'AOX1 region of the chromosome of pichia pastoris strain X33. The X33 cells were made competent as recommended by the manufacturer's instructions, and electroporated using a Biorad GenePulser. Transformants were selected on media containing 100ug/ml Zeocin. Then the protein was induced to express by methanol, and one high expression X33 was chosen by ELASA. The rPSA was purified from the yeast cell culture supernatant and characterized by SDS-PAGE, Western analysis, and was assayed enzyme activity by s-2586. The active, mature rPSA was successfully in Pichia pastoris X33#6 which was chosen. The final yield of active rPSA from Pichia pastoris was 1.2mg/L. The most suitable methanol density to induce express is 1.5%. The rPSA can be purified by cation-exchange chromatography. The rPSA has chymotrypsin-like activity, and condition of the activity determination is 30℃. The chymotrypsin-like activity of rPSA is obvious higher than that of PSA in seminal vesicle fluid of normal male.Large-scale production of active rPSA will be useful in the the study of PSA biology character, clinic detection and therapeutic use and will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. The product can be used to monoclonal antibody, and ELASA assays, which can be reduced price in PCa detection.
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