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/zinc superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reaction (PCR) based technique from cDNA synthesized from black porgy, Acanthopagrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed slightly higher identity (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 required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondria targeting sequence, which suggests that the black porgy cDNA clone encodes a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDNA clone can express active Cu/Zn-SOD enzyme in E. coli.
Lin
C T
CT
Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung, Taiwan. ctlin@ntou66.ntou.edu
Lee
T L
TL
Duan
K J
KJ
Ken
C F
CF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D010473:Perciformes / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000235:genetics*
D018076:DNA, Complementary; D013482:Superoxide Dismutase