title: GST fusion proteins cause false positives during selection of viral movement protein specific single chain antibodies.
abstract: Glutathione S-transferase (GST) fusion proteins are used frequently for investigating protein-protein and protein-DNA interactions. The present study demonstrates that the use of GST fusion proteins caused false positives during selection of phage-displayed single-chain antibody fragments (scFvs) specific for three domains of the movement protein (NS(M)) of tomato spotted wilt virus (TSWV). To identify and exclude the false positives when using GST as a fusion partner linked to the antigen of interest, indirect phage enzyme-linked immunosorbent assay (ELISA) was compared with capture phage ELISA. Of 210 enriched phage clones, indirect phage ELISA identified 106 clones specific for binding to GST-domain fusions but not to GST. In contrast, using capture phage ELISA, all 106 selected clones were identified as false positives, reacting with the GST fusion proteins and GST. This was confirmed by characterization of soluble scFv antibodies. The data indicate that GST fusion proteins seem unsuitable for screening of phage-displayed antibody fragments and it is essential to use capture phage ELISA, instead of the indirect phage ELISA used commonly to exclude false positives in characterization of selected clones with GST fusion proteins.
authors.affiliation: Institut für Biologie I (Botanik/Molekulargenetik), RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: M Y
authors.initials: MY
authors.lastname: Zhang
authors.forename: S
authors.initials: S
authors.lastname: Schillberg
authors.forename: S
authors.initials: S
authors.lastname: Zimmermann
authors.forename: Y C
authors.initials: YC
authors.lastname: Liao
authors.forename: G
authors.initials: G
authors.lastname: Breuer
authors.forename: R
authors.initials: R
authors.lastname: Fischer
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis* / Q000276:immunology; D001435:Bacteriophages; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005189:False Positive Reactions; D005982:Glutathione Transferase / Q000235:genetics* / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000032:analysis / Q000276:immunology; D019151:Peptide Library; D053830:Plant Viral Movement Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012995:Solubility; D017865:Tospovirus / Q000276:immunology*; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D053830:Plant Viral Movement Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase