title: New tags for recombinant protein detection and O-glycosylation reporters.
abstract: Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Petris
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Bestagno
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Arnoldi
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
authors.forename: Oscar R
authors.initials: OR
authors.lastname: Burrone
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D012401:Rotavirus / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; C434828:NSP5 protein, rotavirus group A; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D017361:Viral Nonstructural Proteins