Purification and characterisation of recombinant sea urchin metallothionein expressed in Escherichia coli.
Metallothioneins (MT) are metalloproteins expressed tissue specifically during the development of the sea urchin, Strongylocentrotus pururatus. To explore their structural and functional features and to compare them with those of the evolutionary distant mammalian MTs, one isoform (MTA) was obtained as the cadmium-containing form, from synthetic cDNA heterologously expressed in Escherichia coli. The purified protein was identified as the desired product by a combination of peptide-map analysis, amino acid sequence analysis and ion-spray mass spectroscopy. The existence of seven 113Cd NMR resonances revealed that the recombinant protein binds seven Cd ions/molecule. The position of the NMR resonances (605-695 ppm) and the electronic absorption features suggest that the sea urchin MTA, like the mammalian MTs, possesses tetrahedrally coordinated cadmium-thiolate clusters. With its large Stokes' radius, sea urchin MTA resembles the mammalian forms, suggesting a comparable elongated molecular shape. Measurements by spectrophotometric pH titration of cadmium binding by the recombinant protein suggest that it possesses two metal-thiolate clusters of distinctly different stability. At pH 7 the average apparent association constant for Cd2+ in the clusters is about 20-times weaker in sea urchin MTA than in rabbit MT-2.
Wang
Y
Y
Biochemisches Institut, Universität Zürich, Switzerland.
Mackay
E A
EA
Kurasaki
M
M
Kägi
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D005813:Genes, Synthetic; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008322:Mammals; D008668:Metallothionein / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012617:Sea Urchins / Q000378:metabolism*; D013053:Spectrophotometry
D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein