Chimeras of human extracellular and intracellular superoxide dismutases. Analysis of structure and function of the individual domains.
Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Since EC-SOD is too large a protein for structural determination by multi-dimensional NMR and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. By fusion protein techniques we have previously shown that an amphipathic alpha-helix in the N-terminal domain of hEC-SOD is essential for the tetramer interaction. However, the central domain, which is homologous to intracellular hCuZnSOD, has also been proposed to be involved in the tetramer formation. Despite great efforts, the production of recombinant hEC-SOD in prokaryotic systems or simple eukaryotes (such as yeast) has failed. This lack of success has greatly complicated large-scale production and genetic engineering of the protein. In the study reported here, we constructed two chimeras comprising the N- or the N- and C-terminal domains from hEC-SOD fused to hCuZnSOD, called FusNCZ and PseudoEC-SOD, respectively. We show that these proteins can be produced in large quantities in Escherichia coli, that they can be purified with high yields and that the characteristics of PseudoEC-SOD closely resemble those of hEC-SOD. Further, we extended our studies of the nature of the subunit interaction by investigating the involvement of the central domain.
D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D005109:Extracellular Matrix / Q000737:chemistry*; D015870:Gene Expression; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000737:chemistry*