Molecular Cloning of a cDNA Encoding Copper/Zinc Superoxide Dismutase from Papaya Fruit and Overexpression in Escherichia coli

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• 作者：Ming-Tse Lin ; Ti-Jung Kuo ; and Chi-Tsai Lin
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：1998
• 出版时间：January 1998
• 年：1998
• 卷：46
• 期：1
• 页码：344 - 348
• 全文大小：96K
• 年卷期：v.46,no.1(January 1998)
• ISSN：1520-5118

文摘

A full-length complementary DNA (cDNA) clone encoding a putativecopper/zinc superoxidedismutase (Cu/Zn SOD) of papaya fruit, Carica papaya L. cv.Tainong 2, was amplified accordingto the polymerase chain reaction technique from cDNA synthesized fromfruit messenger RNA.Nucleotide sequence analysis of this cDNA clone revealed that itcomprised a complete open readingframe coding for 152 amino acid residues. The deduced amino acidsequence showed higher identity(81.6-84.9%) with the sequences of the cytosolic Cu/Zn SODs thanthose of the chloroplast fromother plant species, and no recognizable plastid targeting peptide wasfound. These suggest thatthe papaya fruit cDNA clone encodes a cytosolic Cu/Zn SOD. Theresidues required for coordinatingcopper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 andAsp-82), as well as the twocysteines (56 and 145) that form a single disulfide bond, are conservedas they are among all reportedCu/Zn SOD sequences. In addition, the coding region of Cu/Zn SODcDNA from papaya wasintroduced into an expression vetor, pET-20b(+), and transformedinto Escherichia coli AD494(DE3)pLysS. A predominant protein band was detected byCoomassie blue staining of native PAGE,and activity staining confirmed the result of Coomassie blue staining.These indicate that thisCu/Zn SOD cDNA clone can overexpress active Cu/Zn SOD enzyme inE. coli.Keywords: Copper/zinc superoxide dismutase; Cu/Zn SOD; papaya(Carica papaya L. cv. Tainong2)