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Expression and characterization of recombinant human Cu,Zn-superoxide dismutase in Escherichia coli


Expression and characterization of recombinant human Cu,Zn-superoxide dismutase in Escherichia coli



Journal of Biochemistry and Molecular Biology 30(1): 60-65



ISSN/ISBN: 1225-8687

Expression of human Cu.Zn-superoxide dismutase (SOD) with activity comparable to human erythrocyte enzyme was achieved in E. coli B21(DE3) by using the pET-17b expression vector containing a T7 promoter. Recombinant human SOD was found in the cytosol of disrupted bacterial cells and represented gt 25% of the total bacterial proteins. The protein produced by the E. coli cells was purified using a combination of ammonium sulfate precipitation, Sephacryl S-100 gel filtration and DEAE-Sephacel ion exchange chromatography. The recombinant Cu,Zn-SOD and human erythrocyte enzyme were compared using dismutation activity, SDS-PAGE and immunoblotting analysis. The mass of the subunits was determined to be 15,809 by using a electrospray mass spectrometer. The copper specific chelator. diethyldithiocarbamate (DDC) reacted with the recombinant Cu,Zn-SOD. At 50 mu-M and 100 mu-M concentrations of DDC, the dismutation activity was not inhibited for one hour but gradually reduced after one hour. This result suggests that the reaction of DDC with the enzyme occurred in two distinct phases (phase I and phase II). During phase I of this reaction, one DDC reacted with the copper center, with retention of the dismutation activity while the second DDC displaced the copper, with a loss of activity in phase II.

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Accession: 008655696

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