Flag Peptide
Flag peptide is a small hydrophilic tag that can be used to label protein in many different applications. It is a 8 residue sequence that is encoded by a short oligonucleotide sequence and can be added to the N or C terminal of the protein it is attached to. It also contains the enterokinase cleavage site (DDDK) that allows it to be removed from proteins after purification by using the enterokinase enzyme. The FLAG tag is more hydrophilic than other common epitope tags, and therefore less likely to negatively impact the function of the protein to which it is attached.

The FLAG-tag is highly immunogenic and can be easily detected with high specificity by monoclonal antibodies M1 and M2 (also known as anti-flag antibodies). The fusion between the protein and the tag makes it Ca2+ dependent and the complex formed can be broken by the addition of EDTA. It is an excellent choice for affinity chromatography and other immunoprecipitation assays.

Studies have shown that the presence of the FLAG tag does not significantly impact protein crystallization and X-ray diffraction ability. The FLAG peptide sequence can be easily replaced with glutamate without affecting binding to anti-FLAG M2 antibody, indicating that the interacting residues are not essential for binding.

The FLAG peptide is highly charged due to the 11 units of aspartate within its sequence. This gives it the ability to expand its conformation in aqueous solution due to electrostatic repulsive forces between aspartate molecules. This expansion causes it to be able to form salt bridges with other proteins that contain aspartate residues. This feature helps to improve protein purity and efficiency in the purification process.

In the crystal structure of apo anti-FLAG M2 and its complex with the FLAG peptide, the interaction between the two structures is characterized by several hydrogen bonds, salt bridges and ionic interactions (Figure 2, Table 2). It appears that the interaction is mostly driven by the polar side chains of the FLAG peptide. For example, Asp1 forms a salt bridge with the M2 light chain His31 and Asp6 forms a hydrogen bond with the carboxamide side chain of the M2 heavy chain Arg32.

It is interesting to note that the apo anti-FLAG M2 and M2 in complex with the FLAG peptide have markedly different conformational changes than M2 in its native state. In particular, the M2 Fab is positioned closer to the FLAG peptide in the complex than in its apo state. Moreover, the M2 peptide is more tightly wrapped around the M2 Fab in the complex than in the apo state. These observations suggest that the binding of the FLAG peptide to M2 alters the conformation of M2. As such, this molecule may have additional functions that are not yet known. This is an exciting discovery that can help to increase the usefulness of this ligand for various biotechnology applications.