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標題: | 水稻銅鋅超氧歧化?的表達、純化與性質研究 Overexpression, Purification and Characterization of Rice CuZn-Superoxide Dismutase in E. coli |
作者: | Hung-Chi Liu 劉宏基 |
出版年 : | 1997 |
學位: | 碩士 |
摘要: | 本研究室將已選殖之水稻細胞質型銅鋅超氧歧化?(cytosolic CuZnSOD)的cDNA接進pGEX-2T表現載體,且送入大腸桿菌XL1 blue品系中表達。以IPTG誘導大量表現,並經親和層析法純化後,每公升菌液可回收10 - 30 mg的GST-CuZnSOD融合蛋白質。經thrombin作用後,可將GST和CuZnSOD切開,而得到重組型(recombinant)CuZnSOD(rCuZnSOD)。rCuZnSOD較水稻內生的CuZnSOD多兩個胺基酸,此經蛋白質N端序列分析得以證實。GST-CuZnSOD和rCuZnSOD之單元體分子量分別為46 kDa及18.5 kDa,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。其酵素活性也會受過氧化氫及氰化鉀的抑制,但受過氧化氫抑制的程度較水稻內生的為小。由等電焦集電泳分析兩者的pl值皆介於pH 5.2-5.6間。融合蛋白質加熱到60℃以上活性幾乎完全消失;而rCuZnSOD在加熱至100℃後活性仍有30%左右。在酸性(pH<4)環境下,融合蛋白質的活性受到抑制,而在pH 5 -10則較不受影響;rCuZnSOD的活性受pH值影響的程度相對的較小。在0.5 - 2% SDS處理後,融合蛋白質的活性下降,而rCuZnSOD活性受SDS的影響較輕微。融合蛋白質經胰蛋白?或胰凝乳蛋白?部份水解後的產物中,有些仍具有酵素活性。 用製備式膠體電泳進一步純化之融合蛋白質及rCuZnSOD,分別製備抗體。西方墨點法結果顯示二者抗體皆可辨識水稻葉或根粗萃取液中分子量約18.5 kDa及15.7 kDa的蛋白質,此可能是水稻細胞質型和葉綠體型之CuZnSOD的單元體。該抗體對玉米、煙草及菠菜之萃取液亦表現交叉反應,此結果顯示抗水稻細胞質型CuZnSOD的抗體,會辨識其他植物的CuZnSOD。 In our laboratory, the coding region of rice cytosolic CuZnSOD cDNA has inserted into the pGEX-2T expression vector and transformed to E. coli. Transformed E. coli expressed the GST-CuZnSOD fusion protein at levels greater than 20% of soluble protein under optimized conditions, which obtained about 10 ? 30 mg/L bacterial culture. The GST-free recombinant CuZnSOD (rCuZnSOD) can be obtained by thrombin digestion. By SDS-PAGE analysis, the molecular weights of the monomer of GST-CuZnSOD and rCuZnSOD are 46 kDa and 18.5 kDa, respectively. The SOD activity was retained in the dimer of fusion protein or rCuZnSOD. The enzyme activity was inhibited by cyanide; but the rCuZnSOD and fusion protein showed different property from the native form of plant CuZnSOD towards hydrogen peroxide. The pl values of both were estimated between 5.2 ? 5.6 by isoelectric focusing. The fusion protein was unstable at heat treatment, acidic pH, and SDS treatment; but the rCuZnSOD was more stable than fusion protein at similar tests. Some of the partial digested products of fusion protein by trypsin or chymotrypsin still retained SOD activity. The antibodies prepared from the rCuZnSOD or fusion protein respectively, showed the cross-reactivity to the subunit of rice cytosolic CuZnSOD, chloroplastic CuZnSOD, and that of other plants. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76273 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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