Immuno-affinity purification of 2B8-tagged proteins

Abstract

Detection, purification and characterization of proteins are essential procedures in the field of biochemistry. Epitope tag systems are commonly used to characterize unknown proteins. The Deinococcus radiodurans bacterial phytochrome (DrBphP) protein has been used as an antigen to generate anti-DrBphP mouse monoclonal antibodies and to identify their specific epitopes. Among these antibodies, the 2B8 monoclonal antibody recognizes an epitope of 9 amino acids (RDPLPFFPP). The 2B8 epitope does not match the amino acid sequence for any known protein. On Western blot analysis, the 2B8 antibody showed strong and highly specific interactions with the 2B8 epitope. These results suggest that the 2B8 epitope-antibody is a useful epitope tag system for protein characterization. In addition, we generated a modified epitope (RDPLPAFPP) via point mutation in a previous study. This modified epitope showed significantly increased reactivity with the 2B8 antibody. In this study, we developed a protein purification system using the 2B8 epitope tag and antibody. 2B8 antibodies were bound to protein G-agarose beads as affinity ligands. Recombinant DrBphP proteins were then exposed to 2B8 antibody-bound protein G-agarose beads. Bound DrBphP proteins were then eluted by competition with the original or modified 2B8 epitope peptides. DrBphP proteins were successfully purified via an affinity chromatography system using a 2B8 original peptide and even better purified using the 2B8 modified peptide. These findings indicate that the 2B8 epitope tag system is a better tool for protein detection and purification.