Purification and characterization of recombinant Caenorhabditis elegans metallothionein.
The roundworm Caenorhabditis elegans adapted for survival at high concentrations of Cd(II) expresses two isoforms of metallothionein, CeMT-I and CeMT-II. To characterize one of these proteins CeMT-II was prepared as its Cd containing form by expressing its cDNA heterologously in Escherichia coli. The purified 63-amino-acid protein was identified as the desired product by ion-spray mass spectrometry and was found to resemble in most of its chemical and spectroscopic features the metallothioneins of other animal phyla. The recombinant protein contains a total of 18 cysteine residues and, as documented by electrophoresis and mass spectrometry, binds firmly six Cd ions through the cysteine's side chains. The (113)Cd NMR spectrum features six (113)Cd resonances. Their chemical shift positions between 615 and 675 ppm denote the existence of clusters of tetrahedrally coordinated cadmium thiolate complexes. The metal thiolate coordination dominates also the electronic far-UV absorption spectrum. It is characterized by a massive absorption profile with Cd thiolate shoulders at 255 and 235 nm. Upon replacement of Cd by Zn the profile was blue-shifted by 30 nm.
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Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
Mackay
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Gehrig
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Hunziker
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PE
Kägi
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JH
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000737:chemistry* / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D015870:Gene Expression; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D018509:DNA, Helminth; D004274:DNA, Recombinant; D015801:Helminth Proteins; D011994:Recombinant Proteins; D008668:Metallothionein