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題 名 | Over-expression and Characterization of Copper/Zinc-superoxide Dismutase from Rice in Escherichia Coli=水稻銅鋅超氧歧化酶在大腸桿菌中的表達與性質研究 |
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作 者 | 潘素美; 黃冠博; 劉宏基; | 書刊名 | Botanical Bulletin of Academia Sinica |
卷 期 | 40:4 1999.10[民88.10] |
頁 次 | 頁275-281 |
分類號 | 434.113 |
關鍵詞 | 銅鋅超氧歧化酶; 大腸桿菌; 水稻; 超氧歧化酶; CuZnSOD; Escherichia coli; Oryza sativa; Superoxide dismutase; |
語 文 | 英文(English) |
中文摘要 | 以 PCR 合成水稻細胞質型銅錚超氧歧化? (CuZnSOD) 的 cDNA 序列後接進 pGEX-2T 表現載體,且送入大腸桿菌 XL1blue 品系中表達。IPTG 誘導已轉型之大腸桿菌其融合蛋白質表現,其蛋白質產量約占可溶蛋白質之 20% 。並經親和層析法純化後,每公升菌液可回收 10~30mg 的 GST-CuZnSOD 融合蛋白質。 GST-CuZnSOD 融合蛋白質經 thrombin 作用後,可將 GST 切開而得到重組型 CuZnSOD (rCuZnSOD)。 GST-CuZnSOD 和 rCuZnSOD 之單元體分子量分別為 43kDa 及 18kDa,兩者之原態皆以二元體的形式存在;且都保有超氧歧化?的活性。比較融合蛋白質和 rCuZnSOD 的活性對熱、 pH 值和 SDS 處理之效應。二者其酵素活性會受過氧化氫及氰化鉀的抑制 , 但其表現對過氧化氰抑制的抗性。此特性與植物原態CuZn SOD 蛋白質的特性不同。以純化之融合蛋白質製備之抗體,會辨識水稻細胞質型和葉綠體型之CuZnSOD的單元體。並對玉米、煙草及波菜之萃取液亦表現交叉反應。 |
英文摘要 | The over-expression and characterization of cytosolic Cu/Zn-superoxide dismutase (CuZnSOD) from rice in Escherichia coli have been achieved. The full cDNA sequence coding for the rice cytosolic CuZnSOD was made by PCR and inserted into a pGEX-2T expression vector. The recombinant DNA was transformed to E. coli XLI blue. Transformed E. coli expressed GST-CuZnSOD at levels greater than 20% of soluble protein under optimized conditions, and 10-30 mg of fusion protein can be purified from IL bacterial culture by affinity gel. The purified fusion protein was cleaved to remove GST and produced recombinant CuZnSOD (rCuZnSOD). The MWs of a subunit of the fusion protein and rCuZnSOD were 43 kDa and 18 kDa, respectively, as predicted. The SOD activity was retained as the dimer for both forms. The fusion protein and rCuZnSOD were characterized for thermostability and the effects of pH and SDS on its activity by 10% nondenaturing gel. They showed resistance to the inhibition of hydrogen peroxide, in contrast to the native form of the plant CuZnSOD. Antiserum prepared from the GST-CuZnSOD fusion protein showed cross-reactivity to the subunits of rice cytosolic and plastidic CuZnSOD, and to those of other plants. |
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