Genetic design of a gene for human metallothionein II and its expression as an active fusion protein in Escherichia coli

Mitsuo Yamashita, Hidefumi Kuwata, Hidehiko Murakami, Yoshikatsu Murooka

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

A synthetic DNA coding for human metallothionein II (hMT-II) was designed for efficient expression in Escherichia coli and cloned into a vector, pUEX2. Upon induction by heating, the gene for hMT-II was expressed as a fusion protein between β-galactosidase and hMT-II. The fusion protein accounted for approximately 24% of the total protein between β-galactosidase and hMT-II. The fusion protein accounted for approximatedly protein with a molecular mass of approximately 122 kDa was purified in homogeneity by washing the inclusion bodies with a solution of Triton X-100. The insoluble protein was easily solubilized in the presence of 2-mercaptoethanol and sodium dodecyl sulfate without any need for denaturants, such as urea or guanidine hydrochloride. The fused hMT-II protein bound to radioactive cadmium. A competition experiment with radioactive cadmium and various heavy metals showed that the hMT-II fusion protein had a higher affinity for Cu2+, Co2+, and Ag+ ions than Cd2+ and Zn2+ ions. Thus, we succeeded in over expression of hMT-II as an active form in E. coli.

Original languageEnglish
Pages (from-to)113-118
Number of pages6
JournalJournal of Fermentation and Bioengineering
Volume77
Issue number2
DOIs
Publication statusPublished - 1994
Externally publishedYes

Fingerprint

Metallothionein
Escherichia coli
Fusion reactions
Genes
Proteins
Galactosidases
Cadmium
Ions
Mercaptoethanol
Guanidine
Octoxynol
Sodium dodecyl sulfate
Molecular mass
Heavy Metals
Washing
Urea
Sodium Dodecyl Sulfate
Heavy metals
DNA
Heating

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

Genetic design of a gene for human metallothionein II and its expression as an active fusion protein in Escherichia coli. / Yamashita, Mitsuo; Kuwata, Hidefumi; Murakami, Hidehiko; Murooka, Yoshikatsu.

In: Journal of Fermentation and Bioengineering, Vol. 77, No. 2, 1994, p. 113-118.

Research output: Contribution to journalArticle

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