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標題: | 水稻含錳超氧歧化?在細菌中之表達及性質研究 Overexpression and Characterization of Rice Manganese Superoxide Dismutase in E. coli |
作者: | Yin-Chen Tzeng 曾尹貞 |
出版年 : | 1997 |
學位: | 碩士 |
摘要: | 利用PCR的方式,將選殖之水稻含錳超氧歧化?(MnSOD)基因的cDNA序列大量增殖,並接進表現載體pGEX-4T-1中,再送入大腸桿菌DH5α的菌株裡表現。建構好的質體經由Enzyme digestion、Southern、PCR及DNA序列分析後確定接入且無誤。細菌培養後,以0.1mM IPTG誘導GST-MnSOD融合蛋白質大量表現,並經由親和層析純化,得到均質的GST-MnSOD。但GST-MnSOD大部份會形成inclusion body,其可以6M尿素處理及透析後,回收部份GST-MnSOD。 GST-MnSOD經過thrombin處理後,可得到比水稻的含錳超氧歧化?多五個胺基酸的重組型MnSOD(recombinant MnSOD, rMnSOD)。GST-MnSOD及rMnSOD兩種蛋白質,均具有酵素活性,抑制劑H2O2及KCN不會抑制其活性,故兩者仍保有MnSOD的活性。以10% SDS聚丙烯醯胺膠體電泳分析其單元體分子量約各為50kDa、23kDa;以5-20%梯度聚丙烯醯胺膠體電泳的結果,推測兩種蛋白質都以二元體的形式存在。以等電焦集電泳分析GST-MnSOD的pI值介於pH 4.74-4.97之間,而rMnSOD則約為pH 4.64。兩種蛋白質均可耐熱60℃、20分鐘,但於80℃則幾乎不具活性;在鹼性環境中較穩定,而於pH 4.0以下則明顯失去活性。 在探討水稻MnSOD對於T7 RNA polymerase離體轉錄效率的影響時,發現GST蛋白質能提昇RNA產量達24倍之多,SOD卻不會影響轉錄效率。 Mn-superoxide dismutase (MnSOD) had been cloned from rice cDNA library. PCR was used to insert the DNA (mmnsod) coding for the rice mature MnSOD protein into the pGEX-4T-1 expression vector. The recombinant DNA was transformed to Escherichia coli DH5α and the construct was identified by enzyme digestion, southern, PCR and DNA sequence analysis. Expression of GST-MnSOD fusion protein was induced by adding 0.1 mM IPTG to bacterial cultures and the homo-geneous GST-MnSOD was purified by the GST-glutathione affinity system. But most of GST-MnSOD protein in E. coli resulted in an insoluble aggregate which could be recovered by 6M urea and dialysis. Thrombin treatment could cleave GST-MnSOD into GST and rMnSOD (recombinant MnSOD, which had extra five amino acids in N terminal sequence than rice instric MnSOD). Both purified GST-MnSOD and rMnSOD had SOD activity, and were still insensitive to KCN and H2O2, which were characteristics of MnSOD. 10% SDS polyacrylamide gel showed that the monomer molecular weight of the two proteins was 50 kDa and 23 kDa. The native form, estimated by 5-20% gradient polyacrylamide gel electrophoresis, was dimer both in GST-MnSOD and rMnSOD. The isoelectric point of rMnSOD was 4.64, but GST-MnSOD was in the range of pH 4.74 - pH 4.97. The SOD activity declined to 30 % when the two protein incubated at 60℃ for 20 minutes, but more stable at alkaline pH environment. In vitro transcription containing T7 RNA polymerase and [α-35S] CTP are used to estimate the RNA relative products. The results indicated that GST (glutathione-S-transferase) can dramatically enhance the transcription, but SOD showed no this function. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76292 |
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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