Molecular Design of Novel Metal-binding Biomolecules, Oligomeric Metallothioneins Yoshikatsu
Murooka* Michishi Goya Sung-Hye Hong Hisayo Ono Mitsuo Yamashita Noriaki Hirayama *Corresponding Author
Metallothionein (MT) is a suitable model for investigating molecular interactions relating to the handling of metals in the cells. However, the production of functional MT proteins in microorganisms has been limited because of the instability of MT - the thiol group of cysteine is easily oxidized and proteolysis occurred. To increase the binding ability and to stabilize MT protein, we designed genes for dimeric and tetrameric human MT and successful overexpressed in Escherichia coli to generate functional oligomeric MTs. An human MT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Metdeficient hMT-II was ligated to create a dimeric MT, from which a tetrameric MT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. Cells of E. coli carrying the oligomeric MT genes showed resistance toward Zn and Cd toxicity. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apo-metallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four higher than those of the hMT-II monomer protein. These novel oligomeric MTs could be useful as biomaterials, such as detoxification and bioremediation for heavy metals. |
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