Molecular cloning, characterization, and expression of a cDNA coding copper/zinc superoxide dismutase from black porgy

A full-length complementary DNA (cDNA) clone encoding a putative copper/zin c superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reac tion (PCR) based technique from cDNA synthesized from black porgy, Acanthop agrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone reve aled that it comprised a complete open reading frame coding for 154 amino a cid residues. The deduced amino acid sequence showed slightly higher identi ty (72.8-78.1%) with shark and swordfish Cu/Zn-SOD than with Cu/Zn-SOD from mammalian (68.1-70.7%) and plant (55.5-56.5%) sources. The residues requir ed for coordinating copper and zinc are conserved as they are among all rep orted Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondr ia targeting sequence, which suggests that the black porgy cDNA clone encod es a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy w as introduced into an expression vector, pET-20b(+), and transformed into E scherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDN A clone can express active Cu/Zn-SOD enzyme in E. coli.