title: Production of calmodulin-tagged proteins in Drosophila Schneider S2 cells: a novel system for antigen production and phage antibody isolation.
abstract: We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.
authors.affiliation: Department of Haematology, University of Cambridge and the National Blood Service Cambridge, Cambridge, UK.
authors.forename: Nicola S
authors.initials: NS
authors.lastname: Jennings
authors.forename: Peter A
authors.initials: PA
authors.lastname: Smethurst
authors.forename: C Graham
authors.initials: CG
authors.lastname: Knight
authors.forename: Marie N
authors.initials: MN
authors.lastname: O'Connor
authors.forename: Lotta
authors.initials: L
authors.lastname: Joutsi-Korhonen
authors.forename: Prachi
authors.initials: P
authors.lastname: Stafford
authors.forename: Jonathan
authors.initials: J
authors.lastname: Stephens
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Garner
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Harmer
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Watkins
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000235:genetics
chemical_list: D000941:Antigens; D002147:Calmodulin; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins