Cloning and expression of a seed-specific metallothionein-like protein from sesame.
A cDNA clone, SiMT encoding an Ec type of metallothionein (MT)-like protein, was isolated from maturing seeds of sesame (Sesamum indicum L.), and its deduced protein sequence shared 47-65% similarity to other known Ec type of MT-like proteins with three highly conserved cysteine-rich segments. The transcript of SiMT was exclusively accumulated in maturing seeds from two weeks after flowering to the end of seed maturation. The results of a southern blot analysis suggested that one SiMT and one SiMT-like gene were present in the sesame genome. Recombinant SiMT fused with glutathione-S-transferase (GST) was over-expressed in Escherichia coli, and purified to homogeneity by affinity chromatography. Recombinant SiMT released from GST was harvested after factor Xa cleavage. Migration of the recombinant SiMT during SDS-PAGE was accelerated when its binding metal ions were depleted by EDTA. The metal-binding capability of recombinant SiMT was measured by inductively-coupled plasma atomic emission spectrometry. Our results show that the recombinant SiMT could trap zinc or copper ions, but not manganese ions, with a stoichiometric ratio (metal ion/SiMT) of approximately 2.
Chyan
Chia-Lin
CL
Department of Chemistry, National Dong Hwa University, Hwalien, Taiwan.
Lee
Tiger T T
TT
Liu
Chiung-Pin
CP
Yang
Yuan-Chang
YC
Tzen
Jason T C
JT
Chou
Wing-Ming
WM
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D018744:DNA, Plant / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008668:Metallothionein / Q000096:biosynthesis* / Q000737:chemistry; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D018749:RNA, Plant / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000737:chemistry / Q000378:metabolism; D031685:Sesamum / Q000737:chemistry* / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D008670:Metals; D018749:RNA, Plant; D008668:Metallothionein; D005982:Glutathione Transferase