Cdna Cloning And Prokaryotic Expression Of Metallothionein In Pelteobagrus Fulvidraco

Metallothioneins(MTs) are low molecular weight, cysteine-rich, inducible metal-binding proteins found in a variety of organisms. MTs play important biological functions such as protective role against excess reactive heavy metal ions, free radical scavengers and reservoir of essential metals that can be donated to other metalloproteins. Metallothioneins were suggested to be involved in detoxification processes triggered by stress.In this paper, a full-length cDNA sequence encoding MT was cloned by homologous clone and RACE method from Yellow cartfish Pelteobagrus fulvidraco.This cDNA sequence is 381 bp in length, containing a 183 bp open reading frame, which encodes 60 amino acid residues. The deduced amino acid sequence showed more than 80% identity with other known fish MTs. Multiple alignment of amino acid sequences of MTs from different fishes shows that MT of P.fulvidraco possesses the highest identity with that of I.punctatus(91%). The deduced peptide, with a predicted molecular weight of 6080 Da and the content of cysteine is the highest which attain 33.3%, following are Serine(18.3%) and Lysine(11.67%), while no aromatic amino acids or histidine residue was found. MT's Amino Acid Sequence has vertebrate conserved sequence model:Cys-X-Cys, Cys-X-X-Cys and Cys-X-Cys-Cys. Semi-quantitive PCR experiment showed the expression of metallothionein in Yellow cartfish was up regulated sigficantly after stimulation of metal-ion, and MT' expression in liver is higher than other ogans.In this paper, the Yellow cartfish P.fulvidraco MT gene was cloned into prokaryotic expression vector pMAL-c2x, the vector pMAL-c2x has a maIE gene encodes maltose-binding protein(MBP), which was in of the purification of the fusion protein by affinity chromatography subsequently. The recombinant plasmids were successfully introduced into E. coli BL21, SDS-PAGE analysis showed that the fusion protein MBP-MT was successfully expressed by the induction of IPTG. Through analyzing the metal-ion tolerance of MT, the metal-ion Cd2+ and Zn2+ tolerance of E. coli cells expressing MBP-MT fusion protein increased. These results suggest that MT functions in E. coli cells.