The expressed alpha domain of mouse metallothionein-I from Escherichia coli displays independent structure and function.
The alpha domain of mouse metallothionein-I (mMT-I) was expressed in E. coli as a C-terminus of the 26 KD glutathione-S-transferase and purified from the cell lysates. The amino acid composition and molecular weight of the expressed protein are as expected. The metal-binding stoichimetry was determined to show that divalent metals bind to the expressed alpha domain at the desired ratio of 4:1. The ultraviolet absorbance, circular dichroism spectra and the atomic force microscopy indicate that it can form the proper metal-thiolate structure as in the whole MT molecule. The apparent affinity of the expressed alpha domain to bind cadmium is 1.8-fold stronger than the recombinant mMT-I when detected by the reaction with DTNB. The ability to scavenge hydroxyl free radicals remains higher than the whole MT molecule. All the results demonstrate that the expressed alpha-domain from E. coli exhibits independent biochemical and physiological structure/function without the assistance of beta domain.
Xiong
Y
Y
National Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing, China.
Chen
Y
Y
Ru
B
B
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002104:Cadmium / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016166:Free Radical Scavengers / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017665:Hydroxyl Radical / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018625:Microscopy, Atomic Force; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism
D000596:Amino Acids; D003574:Cytochrome c Group; D016166:Free Radical Scavengers; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D017665:Hydroxyl Radical; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid; D015032:Zinc