TY - JOUR
T1 - Molecular design of novel metal-binding oligomeric human metallothioneins
AU - Hong, S. H.
AU - Gohya, M.
AU - Ono, H.
AU - Murakami, H.
AU - Yamashita, M.
AU - Hirayama, N.
AU - Murooka, Y.
N1 - Funding Information:
Acknowledgements This work was supported by a grant-in-aid for scientific research from the Ministry of Culture, Education, Science, and Sports (Monbusho Kiban B, No. 11450315 and Tokutei A, No. 11132246). We thank Drs. Vallee and Maret of Harvard Medical School for technical training and advice for MT characterization.
Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 2000
Y1 - 2000
N2 - 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.
AB - 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.
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U2 - 10.1007/s002530000360
DO - 10.1007/s002530000360
M3 - Article
C2 - 10952009
AN - SCOPUS:0033913468
SN - 0175-7598
VL - 54
SP - 84
EP - 89
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 1
ER -