Molecular design of novel metal-binding oligomeric human metallothioneins |
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| Authors: | S-H Hong M Gohya H Ono H Murakami M Yamashita N Hirayama Y Murooka |
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| Institution: | (1) Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita, Osaka 565-0871, Japan e-mail: murooka@bio.eng.osaka-u.ac.jp Tel.: +81-6-6879-7416 Fax: +81-6-6879-7418, JP;(2) Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama-1, Higashi-Hiroshima 739-8527, Japan, JP;(3) Department of Biological Science and Technology, Tokai University, Nishino, Numazu, Shizuoka 410-32, Japan, JP |
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| Abstract: | 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 apo-metallothioneins 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.
Received: 18 October 1999 / Received last revision: 21 January 2000 / Accepted: 13 February 2000 |
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