| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 98 === For reducing extracellular protease degradation of Pichia pastoris when producing recombinant allergens of Dermatophagoides pteronyssinus used for allergen-specific immunotherapy of oral tolerance, we transformed the constructed vector pPICZαA containing Der p 1＊-Linker- Der p 2 gene into the protease-deficient strain, SMD1168, via electroporation in this study. We selected a transformant which present the highest productivity for further research by using a sandwich ELISA specific to Der p 2. We compared the productivity in Hinton’s flasks between SMD1168 and X-33 in different mediums of BSM and BMGY with addition of protease inhibitors,1 mM PMSF or 1 mM EDTA, or 1% protease substrate casamino acid. 1 mM PMSF could reduce 22% of total protease activity according to protease activity assay and it was found that a protein with correct molecular weight on the result of Western blot specific to Der p 2. For these experiments mentioned above, we concluded that addition of protease inhibitor, PMSF, and protease substrate, casamino acid, could reduce proteolytic degradation and the protein with correct molecular weight could be detected. When cultured in Bioflo 110 bioreactor using BSM with 1% casamino acid and added 0.1 mM PMSF every day after methanol induction, the wet biomass achieved to 241.2 mg/mL and fusion allergen was 54.47 μg/mL at 49th hr. The next, we can deliver a vector containing his gene and increase PMSF addition times to maintain the amount of recombinant protein.
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