Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents.
To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents. We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents. This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu(2+) and Zn(2+). The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein. The enzymes were purified to homogeneity and active enzymes were obtained in high yield. The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy.
Ahl
Ing-Marie
IM
Department of Biomedicine and Surgery, Linköping University, S-581 85 Linköping, Sweden.
Lindberg
Mikael J
MJ
Tibell
Lena A E
LA
D015153:Blotting, Western; D003300:Copper / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry; D013058:Mass Spectrometry; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013054:Spectrophotometry, Atomic; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015032:Zinc / Q000737:chemistry
D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc