水稻超氧歧化酶在大腸桿菌中的表現及生化性質研究

Expression and Characterization of Rice Manganese Superoxide Dismutase in Escherichia coli

本文報告水稻超氧歧化酶（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°C，20分鐘後，SOD活性下降至30%；兩者在鹼性環境中均較穩定。

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°C for 20 minutes; but they were more stable in an alkaline pH environment.