The Availability of a Recombinant Anti-SNAP Antibody in VHH Format Amplifies the Application Flexibility of SNAP-Tagged Proteins

• Published in: BioMed research international 2010-01, Vol.2010 (2010), p.1-7
• Main Authors: Aliprandi, Marisa, Sparacio, Eleonora, Pivetta, Flavia, Ossolengo, Giuseppe, Maestro, Roberta, de Marco, Ario
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibody Specificity; Biological and medical sciences; Blotting, Western; DNA repair; Escherichia coli; Fluorescent Antibody Technique; Genetic Vectors; HEK293 Cells; HeLa Cells; Humans; Immunoglobulin G - genetics; Immunoglobulin Heavy Chains - chemistry; Immunoglobulin Heavy Chains - genetics; Labeling; Medical sciences; O-Methylguanine-DNA Methyltransferase - chemistry; O-Methylguanine-DNA Methyltransferase - immunology; O-Methylguanine-DNA Methyltransferase - metabolism; Peptide Library; Pharmacology. Drug treatments; Proteins; Rabbits; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; Staining and Labeling; Twist-Related Protein 1 - immunology
• ISSN: 1110-7243; 2314-6133; 1110-7251; 2314-6141
• DOI: 10.1155/2010/658954

Antibodies are indispensable reagents in basic research, and those raised against tags constitute a useful tool for the evaluation of the biochemistry and biology of novel proteins. In this paper, we describe the isolation and characterization of a single-domain recombinant antibody (VHH) specific for the SNAP-tag, using Twist2 as a test-protein. The antibody was efficient in western blot, immunoprecipitation, immunopurification, and immunofluorescence. The sequence corresponding to the anti-SNAP has been subcloned for large-scale expression in vectors that allow its fusion to either a 6xHis-tag or the Fc domain of rabbit IgG2 taking advantage of a new plasmid that was specifically designed for VHH antibodies. The two different fusion antibodies were compared in immunopurification and immunofluorescence experiments, and the recombinant protein SNAP-Twist2 was accurately identified by the anti-SNAP Fc-VHH construct in the nuclear/nucleolar subcellular compartment. Furthermore, such localization was confirmed by direct Twist2 identification by means of anti-Twisit2 VHH antibodies recovered after panning of the same naïve phage display library used to isolate the anti-SNAP binders. Our successful localization of Twist2 protein using the SNAP-tag-based approach and the anti-Twist2-specific recombinant single-domain antibodies opens new research possibilities in this field.