TITLE	Over-expression and characterization of copper/zinc-superoxide dismutase from rice in Escherichia coli
AUTHOR	Shu-Mei Pan Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China Guan-Bor Hwang Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China Hung-Chi Liu Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China
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ABSTRACT	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 XL1 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 1L 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.
KEYWORD	CuZnSOD; Escherichia coli; Oryza sativa; Superoxide dismutase;
ARTICLE INFO	Botanical Bulletin of Academia Sinica, Volume 40 Number 4 October 1999, page 275-281, 7 pages
PUBLISHER	Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China