Open AccessBiochemical and structural characterization of recombinant copper‐metallothionein from Saccharomyces cerevisiae
Author(s) -
Sayers Zehra,
Brouillon Patricia,
Svergun Dimitri I.,
Zielenkiewicz Piotr,
Koch Michel H. J.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00451.x
Subject(s) - size exclusion chromatography , metallothionein , chemistry , radius of gyration , recombinant dna , copper , chromatography , saccharomyces cerevisiae , escherichia coli , biochemistry , enzyme , polymer , yeast , organic chemistry , gene
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].