Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents

Protein Expression and Purification
Ing-Marie AhlLena Tibell

Abstract

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.

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Citations

Oct 19, 2012·The Journal of Biological Chemistry·Hugo M BotelhoCláudio M Gomes
Aug 24, 2007·Proceedings of the National Academy of Sciences of the United States of America·Per ZetterströmStefan L Marklund
Oct 16, 2014·Proceedings of the National Academy of Sciences of the United States of America·Ashley J PrattElizabeth D Getzoff
Jun 6, 2009·Proceedings of the National Academy of Sciences of the United States of America·Anna NordlundMikael Oliveberg
Sep 21, 2013·International Journal of Molecular Sciences·Joana S CristóvãoCláudio M Gomes
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Apr 28, 2021·Critical Reviews in Food Science and Nutrition·Mohammad Nazmul IslamMohammad S Mubarak

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