FLAG Tag Explained- Epitope Tag for Protein Purification

flag tag epitope for protein purification

FLAG tag (DYKDDDDK) is an epitope tag widely adopted for its high specificity and purity in protein purification. Anecdotally, it is commonly used as a follow-on or alternative to His tag when high purity is desired. This blog post outlines FLAG’s benefits and shortcomings and compares its performance to TriAltus’ CL7 tag.

FLAG tag

FLAG is a short, 8 amino acid sequence DYKDDDDK about 1 kDa in size (D = aspartic acid, Y = tyrosine, K = lysine). Due to its small size, some researchers may choose to leave the tag intact, but cleavage can be performed with enterokinase. Enterokinase cleaves at the recognition sequence Asp-Asp-Asp-Asp-Lys-X, with X being any amino acid other than proline, at the C-terminal end of the lysine. Since this sequence is contained within FLAG’s sequence, enterokinase is commonly used for FLAG cleavage. 

Several FLAG sequences can be used in tandem in a vector to increase the likelihood of antibody binding. FLAG is versatile and can be used either C-terminal or N-terminal to the fusion partner. 

FLAG antibodies

Affinity purification with the FLAG system relies on the relationship between proteins with a FLAG sequence and anti-FLAG antibodies that are cross-linked to agarose resin. The Kd of FLAG and anti-FLAG is around 100 nM, and its specificity serves as an advantage over the non-specific binding of His-tagged proteins to metal ion columns. Protein purity from FLAG is typically >90%. However, the protein binding capacity of anti-FLAG resin columns is quite low with only >0.6 mg protein/mL resin bound. This severely limits the potential yield of a run, especially with larger proteins. 

Eluting FLAG-tagged proteins

Several options exist for eluting FLAG-tagged proteins. Because the binding of FLAG and its antibody is Ca2+ dependent, EDTA can be used to elute tagged protein from the column. Alternatively, FLAG peptides or glycine-HCl pH 3 solutions can be used. Anti-FLAG M1 resin used for purification can be reused up to 20x if periplasmic cell extracts are applied to the column but only up to 3x if crude lysate is applied to the column. 

CL7 vs. FLAG

CL7 offers benefits in purification where FLAG falls short. CL7 and its binding partner Im7 have a Kd of 10^-14- 10^-17 which offers even higher specificity than the affinity between FLAG and its antibodies. Im7 is CL7’s natural immunity binding partner and as such shows no off-target between the two units.

One of the key advantages over FLAG is that Im7 resin columns have a much higher binding capacity of 35-40 mg/mL. This feature in combination with the >100x reusability of Im7 resin drives down the price per use of the system’s materials. 

Additionally, due to the high binding affinity between CL7 and Im7 the system is resistant to high salt buffers. Loading in high salt helps to eliminate impurities earlier in the purification process, resulting in a more pure final protein. CL7/Im7 has a track record of producing proteins in the 97-100% purity range. 

cl7 vs flag tag

FLAG remains a strong option for protein purification in cases where purity and not yield is the priority. For situations in which both are desired, the CL7 system can serve as a one-step solution for protein purification.