Abstract

The corrected cDNA coding for rice mature MnSOD protein was made by PCR and inserted into pGEX-4T-1 expression vector. The recombinant DNA was transformed to E. coli BL21 and expression of GST-MnSOD fusion protein was induced by addition of IPTG to bacterial cultures. The homogeneous GST-MnSOD was purified by the one-step GST-glutathione affinity system. Both purified GST-MnSOD and recombinant MnSOD (rMnSOD) remained MnSOD activity, which showed an insensitivity towards KCN and H2O2. The molecular size of monomer of GST-MnSOD or rMnSOD was 50 kDa and 23 kDa respectively, and the functional form of both was dimer. The isoelectric point of rMnSOD was 4.64, but that of GST-MnSOD was in the range of pH 4.74 - 4.97. The SOD activities of GST-MnSOD and rMnSOD declined to 30 % after incubation at 60 ℃ for 20 minutes; but they were more stable in an alkaline pH environment.

中文摘要

本文報告水稻超氧歧化 (MnSOD) 在大腸桿菌中的表現及生化性質的分析。利用 PCR 將對應到水稻 MnSOD 成熟蛋白質之正確 cDNA 序列接入 pGEX-4T-1 表現載體中，將建構好的質體再轉型至大腸桿菌 BL21 品系中。融合蛋白質 GST-MnSOD 可被 IPTG 誘導表現，而經由 glutathione 親和膠體層析可純化均質之 GST-MnSOD 融合蛋白質。 重組之 MnSOD (rMnSOD) 及 GST-MnSOD 均對 KCN 和 H2O2 不敏感，此即保有生物中原態 MnSOD 的一特性。兩者的單體分子量分別為23 kDa 和 50 kDa，而在原態上均以二元體的形式存在。rMnSOD 的等電點為 4.64，GST-MnSOD 的等電點則介於 pH 4.74－4.975 之間。rMnSOD 及 GST-MnSOD 兩蛋白質處理 60℃， 20分鐘後，SOD 活性下降至 30%；兩者在鹼性環境中均較穩定。 關鍵詞：超氧歧化，含錳超氧歧化，水稻，大腸桿菌。

Keyword: Superoxide dismutase (SOD), MnSOD, Oryza sativa, E. coli.