title: Rational design and expression of a heparin-targeted human superoxide dismutase.
abstract: In order to improve the therapeutic effectiveness of human Cu,Zn superoxide dismutase (HSOD) by targeting it to cell surfaces and increasing its circulatory half-life, we have designed and expressed a heparin-binding derivative of HSOD. This design was based on the idea that structurally independent protein units, HSOD and the heparin-binding A+ helix from protein C inhibitor, could be combined with a carefully chosen linker, GlyProGly, to form a stable, bifunctional protein. The chimeric HSOD-GlyProGly-A+ protein was expressed and secreted to the periplasm of E. coli and had normal SOD activity. HSOD-GlyProGly-A+ had a significantly increased retention time relative to wild-type HSOD on a heparin affinity column, indicating that it was successfully targeted to heparin, and this binding was maintained at physiological ionic strength. When administered to mice, HSOD-GlyProGly-A+ had a half-life of approximately 15 minutes, twice that of wild-type HSOD. Our rational design approach should be generally applicable to the creation of bifunctional chimeric molecules.
authors.affiliation: Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
authors.forename: M
authors.initials: M
authors.lastname: Boissinot
authors.forename: L A
authors.initials: LA
authors.lastname: Kuhn
authors.forename: P
authors.initials: P
authors.lastname: Lee
authors.forename: C L
authors.initials: CL
authors.lastname: Fisher
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: R A
authors.initials: RA
authors.lastname: Hallewell
authors.forename: J A
authors.initials: JA
authors.lastname: Tainer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D015195:Drug Design; D006207:Half-Life; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D006493:Heparin; D013482:Superoxide Dismutase