Pichia pastoris  生產重組塵蟎過敏原之胞外蛋白酶活性 抑制研究

Abstract

為降低 Pichia pastoris 胞外蛋白酶在生產時對重組塵蟎過敏原蛋白的降解，以使用歐洲塵蟎重組過敏原蛋白質進行過敏源專一性的口服耐受性免疫試驗，本研究將先前本實驗室已建構完成含有 Der p 1＊-Linker- Der p 2 基因之 pPICzαA 載體，電轉形進入蛋白酶缺陷株 P. pastoris SMD1168，並利用對 Der p 2 專一性之三明治酵素連結免疫分析法，挑選出表現量最高的轉形株。將重組 P. pastoris SMD1168和重組 X-33 (對照組) 在 Hinton 氏搖瓶中分別以 Base salt medium (BSM) 或 Buffered glycerol complex-medium (BMGY) 培養，並添加蛋白酶抑制劑: 1 mM phenylmethanesulfonylfluoride (PMSF)、1 mM ethylenediaminetetraacetic acid (EDTA)，或是 1% 蛋白酶受質 casamino acid，在這些不同條件下比較。由蛋白酶活性測試可知，添加 1 mM PMSF 可抑制 22% 總蛋白酶活性，且以專一於 Der p 2 之西方點墨法結果可發現正確分子量的重組蛋白質，由以上實驗顯示添加蛋白酶抑制劑PMSF 及蛋白酶受質如 casamino acid 可有效減緩蛋白酶降解現象並偵測到正確分子量之蛋白質。在 Bioflo 110 生物反應器中以 BSM 培養並添加 1% casamino acid且自甲醇誘導後每天添加 0.1 mM PMSF，濕重最高可達 241.2 g/L，融合過敏原表現量於49 小時達54.5 μg/mL。將來可再送入含有合成組胺酸基因的載體至SMD1168並增加 PMSF 添加量，以期維持重組蛋白質之產量。

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.