The FLAG® system utilizes a short, hydrophilic 8-amino acid peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) which is fused to the recombinant protein of interest when expressed from a pFLAG vector. The FLAG peptide includes the binding site for several highly specific ANTI-FLAG monoclonal antibodies (M1, M2, M5) and polyclonal antibodies and conjugates, each with different recognition and binding characteristics. The FLAG peptide is likely to be located on the surface of a fusion protein because of its hydrophilic nature. As a result, the FLAG peptide is more likely to be accessible to antibodies and to cleavage by enterokinase (Ek). In addition, because of the small size of the FLAG® peptide tag, it is not likely to obscure other epitopes, domains, or alter function, secretion, or transport of the fusion protein.
The 3X FLAG system improves upon the original system by fusing 3 tandem FLAG epitopes to a recombinant protein (Fig. 1). A 10-20 fold increased detection enhancement has been shown using 3X FLAG fusions (Fig. 2, 3). As with the original FLAG® tag, it is hydrophilic, contains an enterokinase cleavage site and is relatively small (22 amino acids), therefore, the risk of altering protein function, blocking other epitopes or decreasing the solubility is minimized.