Molecular design of novel metal-binding oligomeric human metallothioneins.
Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs. An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apometallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The oligomeric hMT proteins incubated with Cd ions showed a typical Cd-thiolate absorbance peak at 245-255 nm. The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein. These novel oligomeric hMTs may be useful in bioremediation for heavy metals.
Hong
S H
SH
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Gohya
M
M
Ono
H
H
Murakami
H
H
Yamashita
M
M
Hirayama
N
N
Murooka
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D017931:DNA Primers; D006801:Humans; D008668:Metallothionein / Q000737:chemistry*; D008969:Molecular Sequence Data
D001704:Biopolymers; D017931:DNA Primers; D008668:Metallothionein