Analysis of recombinant human prostasin carrying a serine active site mutation
Prostate cancer is the second leading cause of cancer-related death, and the most commonly diagnosed type of cancer in men. By conventional paradigm, serine proteases have been shown to play a significant role in cancer cell invasion. Prostasin is a serine protease found in abundance in prostate epithelial cells, and also in other tissues and cell types in the body. Prostasin is absent in prostate cancer cell lines. When re-expressed in these prostate cancer cell lines, the invasiveness was decreased. The structural domain responsible for the anti-invasion activity exhibited by prostasin has not yet been characterized. Through site-directed mutagenesis of the serine active residue to an alanine residue, the role that the serine active site plays in anti- invasion can be explored. The cDNA clone of the mutated prostasin was transfected into the 293-EBNA cell line and cultured (293/ProM). The alanine-prostasin mutant, Ala-Pro, was partially purified and confirmed by Western blotting. Additionally, a binding assay was performed using mPBP (mouse prostasin-binding protein), a serpin class serine protease inhibitor. By way of immunoblotting, it was shown that normal wild-type prostasin forms an 82-kDa complex with the mPBP; however, the mutated prostasin does not. In future studies the invasiveness of prostate cancer cells transfected with the mutant prostasin cDNA can be investigated to determine whether prostasin's serine protease activity provides a mechanism for anti-invasion.
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Chai, Karl X.
Bachelor of Science (B.S.)
College of Health and Public Affairs
Molecular Biology and Microbiology
Dissertations, Academic -- Health and Public Affairs;Health and Public Affairs -- Dissertations, Academic;Prostate -- Cancer;Serine proteinases
Length of Campus-only Access
Honors in the Major Thesis
Marcello, Matthew R., "Analysis of recombinant human prostasin carrying a serine active site mutation" (2003). HIM 1990-2015. 336.