Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae.
Methods were developed for large-scale purification of recombinant Cu-metallothionein (Cu-MT) for structural investigations and the determination of Cu-binding stoichiometry. Cu-MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed-phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 +/- 2 Cu(I) per molecule of protein. Small angle X-ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEBS Lett. 379, 85-93].
European Molecular Biology Laboratory, Hamburg, Germany.
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Sayers
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Brouillon
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Svergun
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Zielenkiewicz
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Koch
D002352:Carrier Proteins; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D012542:Scattering, Radiation; D014965:X-Rays
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; C007121:copper thionein; D008668:Metallothionein