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

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Abstract
A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn SOD) of papaya fruit, Carica papaya L. cv. Tainong 2, was amplified according to the polymerase chain reaction technique from cDNA synthesized from fruit messenger RNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequence showed higher identity (81.6-84.9%) with the sequences of the cytosolic Cu/Zn SODs than those of the chloroplast from other plant species, and no recognizable plastid targeting peptide was found. These suggest that the papaya fruit cDNA clone encodes a cytosolic Cu/Zn SOD. The residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, are conserved as they are among all reported Cu/Zn SOD sequences. In addition, the coding region of Cu/Zn SOD cDNA from papaya was introduced into an expression vetor, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant protein band was detected by Coomassie blue staining of native PAGE, and activity staining confirmed the result of Coomassie blue staining. These indicate that this Cu/Zn SOD cDNA clone can overexpress active Cu/Zn SOD enzyme in E. coli.