New tags for recombinant protein detection and O-glycosylation reporters

• Published in: PloS one 2014-05, Vol.9 (5), p.e96700
• Main Authors: Petris, Gianluca, Bestagno, Marco, Arnoldi, Francesca, Burrone, Oscar R
• Format: Article
• Language: English
• Subjects: Advantages; Amino Acid Sequence; Amino acids; Animals; Antibodies, Monoclonal - immunology; Antigenic determinants; Antigens; Biology and Life Sciences; Biotechnology; Chromatography; Cloning; Epitope Mapping; Epitopes - chemistry; Epitopes - immunology; Genetic engineering; Genomes; Glycosylation; Golgi apparatus; HEK293 Cells; Humans; Immunoglobulins; Journalists; Laboratories; Medicine and Health Sciences; Membrane proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Monoclonal antibodies; Peptides; Phosphorylation; Proteins; Recombinant proteins; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Recombinant Proteins - immunology; Research and Analysis Methods; Rotavirus; Rotavirus - metabolism; Scientific research; Single-Chain Antibodies - immunology; Tagging; Tags; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - immunology; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0096700

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.