利用熱休克蛋白質5'端非轉譯區片段調控免疫調節蛋白質GMI於米麴菌表達系統之產量

Abstract

本研究針對提升小孢子靈芝 (Ganoderma microsporum) 之真菌免疫調節蛋白質GMI於米麴菌 (Aspergillus oryzae) 異源蛋白質表達系統之產量及其蛋白質立體結構進行探討。 使用導入A. oryzae同源熱休克蛋白質基因hsp12之5’端非轉譯區策略，可發現於將溫度提升至37oC 及鹽濃度提升至0.7 M NaCl兩種培養條件下，胞外GMI產量顯著增加。但提升鹽濃度亦可促進不含hsp12之5’端非轉譯區對照組之GMI產量，顯示在高鹽濃度處理下，可能有其他機制參與調控。由胞內外GMI相對含量比較可發現，hsp12之5’端在逆境下對GMI分泌至胞外效力影響甚鉅，其可能機制除協助GMI運送定位外，亦可協助mRNA及GMI於轉譯至定位過程中穩定存在。 利用巴斯德畢赤氏酵母菌 (Pichia pastoris) 生產重組蛋白質rGMI，在10 mg/mL濃度時，在20% PEG 4000, 0.6 M NaCl, 0.1 M NaMES pH 6.5條件下，於室溫培養20天可得六角柱狀結晶。利用X-ray繞射方式進行其結構探討，rGMI晶體屬於I4122空間群，三軸長分別為a = b = 95.73, c = 80.07 Å，夾角為

This study was to improve GMI, a fungal immunomodulatory protein from Ganoderma microsporum, production in Aspergillus oryzae heterologous protein expression system. To better understand the function-structure relationship, we also worked on GMI protein structure. A 5’ untranslated region (5’UTR) from A. oryzae homologous heat shock protein 12 gene (hsp12) was used to increase GMI production in A. oryzae. The results suggested that extracellular GMI production significantly increased at 37oC in comparison to that at 30oC, or under stress of 0.7 M NaCl. However, 0.7 M NaCl treatment also enhanced the GMI production of the construct without hsp12 5’UTR, indicating that the enhancement of GMI production under osmotic stress treatment might involve other mechanisms. The results also show hsp12 5’UTR might increase the secretion efficiency of GMI by assisting GMI mRNA stablilization and subcellular location. 10 mg/mL recombinant GMI (rGMI) produced by Pichia pastoris was crystallized by the sitting-drop vapor-diffusion method under the condition of 20% PEG 4000, 0.6 M NaCl, 0.1 M Na MES, pH 6.5. The crystal diffracted X-rays to 2.0 Å resolution using synchrotron radiation and belong to space group I4122, with unit-cell parameter a = b = 95.73, c = 80.07 Å. rGMI is a homotetramer, tetramerized by hydrophobic interaction (T101-I106), and dimerized by charge-charge interaction (T4-K46) and hydrophobic interaction (T4-D98, I7-L17, I7-F19, A11-Y21, W12-Q109, V14-F19).