Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast)

BACKGROUND. Prostate-specific antigen (PSA), a member of the kallikrein fam ily of serine proteases, is a chymotrypsin-like glycoprotein produced by th e prostate epithelium. Elevated serum PSA (>4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA ove r time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular mat rix glycoproteins, as well as cleavage of IGF binding protein-3, a modulato r of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective so urce of enzymatically active protein. Previous efforts to express recombina nt PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expr ession of active recombinant mature PSA in yeast. METHODS. Stable chromosomal integration of a construct consisting of the ye ast cc-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture super natant to homogeneity by strong cation-exchange chromatography, and charact erized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-gly canase digestion, N-terminal amino acid sequencing, and inactivation by a P SA-specific inhibitor. RESULTS. We report the production of active, mature rPSA in Pichia pastoris . Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 mu mol/min mg) as determined using a chromogenic substrate. CONCLUSIONS. Large-scale production of active rPSA will be useful in the ex ploration of PSA effects on tumor cell proliferation, migration and metasta sis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing. Pr ostate 46:298-306, 2001. (C) 2001 Wiley-Liss, Inc.