Molecular cloning of a cDNA coding for copper/zinc superoxide dismutase from zebrafish and its expression in Escherichia coli

摘要

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 zebrafish (Danio rerio) 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 higher identity (73.5-74.3%) with swordfish and shark Cu/Zn-SOD than with Cu/Zn-SOD from mammals (69.6-70.9%) and plants (55.8-56.2%). The amino acid residues required for coordinating copper and zinc are conserved, as they are present in all reported Cu/Zn-SOD sequences. It lacks a targeting sequence, which suggests that the zebrafish cDNA clone encodes a cytosolic Cu/Zn-SOD. Furthermore, the coding region of Cu/Zn-SOD from zebrafish was introduced into an expression vector, pET-23a(+)-thioredoxin, and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE, and the expression pattern was shown by Coomassie blue staining of SDS-PAGE. This indicates that the Cu/Zn-SOD cDNA clone can be expressed in E. coli. [References: 20]