Study On Yeast Expression And Metal-binding Properties Of A Type2Metallothionein(PutMT2)from Puccinellia Tenuifolra

Metallothioneins (MTs) are a family of low molecular mass, cysteine-rich and metal-binding proteins ubiquitous present in plants, animals, fungi and cyanobacteria. Plants MTs are known to be involved in metal detoxification or homeostasis, as they are able to bind metal ions through the thiol-groups of their cysteine residues. Plant MTs are also involved in the scavenging of reactive oxygen species (ROS) and stress responses. The interplay between these roles is not entirely clear, and no specific role for any of them has been assigned.A type2metallothionein gene PutMT2(GenBank, Accession the No. JQ279061) was identified and obtained from yeast cDNA library of Puccinellia tenuifolra screening in the NaCl stress medium. The gene has open reading frame (ORF) of246bp, encoding81amino acids. PutMT2protein was analyzed and predicted by bioinformatics method. Results show that PutMT2owes the molecular weight of about8kDa, and isoelectric point of5.66. The serine and threonine in the middle region are phosphorylation sites, and random coil is the main components of its secondary structure.PutMT2gene expression in yeast improve cells Cd2+, Zn2+, Cr6+, Ag+, Fe2+, Fe3+, NaCl and H2O2tolerance. PutMT2cells are more sensitive to Mn2+, Co2+, Cu2+and Ni2+than control cells. Moreover, Na, Zn, Cu and Fe content of cell metal accumulation and protein binding from control cells and PutMT2yeast cells were determined by atomic absorption spectrometry. The results show that PutMT2can bind Na under certain conditions, and its expression of the yeast makes intracellular Na content decrease. PutMT2protein can bind Zn ion, which promote Zn accumulation in yeast cells in low concentrations of Zn and reduce intracellular Zn content in stress concentration. PutMT2expression in yeast reduced the intracellular copper content. PutMT2Cu-binding ability may affect intracellular Cu ion homeostasis, so that yeast became sensitivity to Cu2+. The expression of PutMT2endowed resistance in yeast to Fe maybe through PutMT2protein binding Fe.PutMT2was overexpressed in Escherichia coli BL21as a fusion protein (GST-PutMT2). A large amount of GST-PutMT2fusion protein is easy to be purified from E. coli BL21efficient expression system. The zinc, copper and sodium addition do not affect the purity of the fusion protein, and adding the appropriate ion concentration will increase the yield of the purified protein.The metal-binding ability of GST-PutMT2fusion protein was analyzed and compared with GST protein by ICP-AES. Results show that PutMT2can bind Cr, Cd, Co, Ag, Ba, Pb, Mn, Zn, Fe, Cu, P, Al and Mg, but maybe not combination with Ni and As. There is no obvious evidence to show PutMT2specific or selective binding tendency of metal ions. The content of Cu, Zn or Fe of PutMT2protein don’t increase with the additional metal concentration increased, which means that PutMT2involved in metal ion homeostasis. Under normal culture conditions, PutMT2can combine with Na. After other metal ions such as Cu, Zn or Fe content increased, is no longer the Na-binding.