title: Bacterial expression and membrane targeting of the rat complement regulator Crry: a new model anticomplement therapeutic.
abstract: Inappropriate or unregulated activation of complement can contribute to pathology in inflammatory diseases. Previous studies have shown that soluble recombinant regulators of complement are effective in animal models and some human diseases. However, limitations include cost, rapid clearance, and unwanted systemic effects. To avoid some of these problems, bacterial expression of regulators has been optimized and methods for the addition of a membrane-targeting moiety to the complement regulator developed. When administered directly to sites of inflammation, membrane-targeted human regulators are retained and inhibit complement-activation locally. To test the efficacy of membrane-targeted complement regulators in vivo, we have undertaken the expression and membrane targeting of the rat-complement regulator Crry. A soluble recombinant form of Crry, containing only the first four short consensus repeats, was expressed in a mammalian expression system and shown to be functional as a fluid phase regulator. To generate the quantities required for testing in vivo, Crry was expressed in bacteria and refolded successfully. Refolded protein had full-complement regulatory activity in vitro. Attachment of a membrane address tag conferred membrane-binding capacity and greatly increased complement regulatory function in vitro. This novel anticomplement agent can now be applied to rat models of arthritis and other inflammatory diseases.
authors.affiliation: Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff CF14 4XX, UK.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Fraser
authors.forename: Claire L
authors.initials: CL
authors.lastname: Harris
authors.forename: Richard A G
authors.initials: RA
authors.lastname: Smith
authors.forename: B Paul
authors.initials: BP
authors.lastname: Morgan
mesh_terms: D000818:Animals; D000954:Antigens, Surface; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D003165:Complement System Proteins / Q000378:metabolism; D006224:Cricetinae; D004926:Escherichia coli; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D011956:Receptors, Cell Surface; D011951:Receptors, Complement / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000954:Antigens, Surface; C513105:Cr1l protein, rat; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011951:Receptors, Complement; D011994:Recombinant Proteins; D003165:Complement System Proteins