Genetic design of stable metal-binding biomolecules, oligomeric metallothioneins

Y. Murooka, M. Toyama, S. H. Hong, M. Gohya, H. Ono, Mitsuo Yamashita, N. Hirayama

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Metallothionein (MT) is a suitable model for investigating molecular interactions relating to the handling of metals in 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 occurs. To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MT and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs. A human MT-II (hMT-II) synthesized with prokaryotic codons, a linker encoding a glycine tripeptide, and Met-deficient 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-MTs were reconstituted with Cd or Zn ions under reducing conditions. The dimeric and tetrameric MT 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 stable oligomeric MTs have potential as a biomaterial for uses such as detoxification and bioremediation for heavy metals.

Original languageEnglish
Pages (from-to)399-412
Number of pages14
JournalBiocatalysis and Biotransformation
Volume19
Issue number5-6
Publication statusPublished - 2001
Externally publishedYes

Fingerprint

Metallothionein
Biomolecules
Metals
Escherichia coli
Proteins
Genes
Proteolysis
Detoxification
Molecular interactions
Bioremediation
Dithiothreitol
Biocompatible Materials
Heavy Metals
Sulfhydryl Compounds
Biomaterials
Microorganisms
Glycine
Heavy metals
Cysteine
Toxicity

Keywords

  • Cd
  • Human metallothionein-II
  • Metal tolerance
  • Overproduction
  • Stabilization
  • Zn

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Catalysis

Cite this

Murooka, Y., Toyama, M., Hong, S. H., Gohya, M., Ono, H., Yamashita, M., & Hirayama, N. (2001). Genetic design of stable metal-binding biomolecules, oligomeric metallothioneins. Biocatalysis and Biotransformation, 19(5-6), 399-412.

Genetic design of stable metal-binding biomolecules, oligomeric metallothioneins. / Murooka, Y.; Toyama, M.; Hong, S. H.; Gohya, M.; Ono, H.; Yamashita, Mitsuo; Hirayama, N.

In: Biocatalysis and Biotransformation, Vol. 19, No. 5-6, 2001, p. 399-412.

Research output: Contribution to journalArticle

Murooka, Y, Toyama, M, Hong, SH, Gohya, M, Ono, H, Yamashita, M & Hirayama, N 2001, 'Genetic design of stable metal-binding biomolecules, oligomeric metallothioneins', Biocatalysis and Biotransformation, vol. 19, no. 5-6, pp. 399-412.
Murooka, Y. ; Toyama, M. ; Hong, S. H. ; Gohya, M. ; Ono, H. ; Yamashita, Mitsuo ; Hirayama, N. / Genetic design of stable metal-binding biomolecules, oligomeric metallothioneins. In: Biocatalysis and Biotransformation. 2001 ; Vol. 19, No. 5-6. pp. 399-412.
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