title: Efficient production of complement (C3d)3 fusion proteins using the baculovirus expression vector system.
abstract: Proteins fused to activated complement (C) fragments elicit enhanced immunogenicity. This "natural adjuvant" effect may have important implications when considering novel vaccination approaches. Here we describe both the construction of a novel fusion protein, consisting of a well characterized test antigen fused to multiple copies of the activated complement component (C3d)3, as well as an efficient method for its expression and production in insect cells. Using the inherent biological advantages of the baculovirus expression system, as well as applying specific infection and harvesting modifications, we have optimized the efficiency of protein production. Our modifications allow purification of fusion proteins directly from cell supernatant in a single anion exchange chromatographic step. This alleviates the requirement for the inclusion of protein affinity tags. The integrity of the purified recombinant protein was evaluated by SDS PAGE analysis, reactivity with antibodies, as well as in vivo by administration as an immunogen.
authors.affiliation: Institute for Immunology and Infection Research, The School of Biological Sciences, University of Edinburgh, Ashworth Laboratories, West Mains Road, Edinburgh EH9 3JT, UK.
authors.forename: Denise V
authors.initials: DV
authors.lastname: Barrault
authors.forename: Michael
authors.initials: M
authors.lastname: Steward
authors.forename: Vivienne F
authors.initials: VF
authors.lastname: Cox
authors.forename: Richard A G
authors.initials: RA
authors.lastname: Smith
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Knight
mesh_terms: D000276:Adjuvants, Immunologic / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D003017:Clostridium tetani / Q000235:genetics / Q000276:immunology; D015933:Complement C3d / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors; D051379:Mice; D008810:Mice, Inbred C57BL; D009036:Moths / Q000235:genetics / Q000276:immunology; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D018411:Spodoptera / Q000235:genetics / Q000276:immunology; D013744:Tetanus Toxin / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D015933:Complement C3d