Regulation of an immunomodulatory protein, GMI, expression in Aspergillus oryzae by introducing a heat shock protein 5’untranslated region

• Main Authors: Ming-yueh Wu, 吳明玥
• Other Authors: Ching-Tsan Huang
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/37148231255180887583

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 96 === 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).