FLAG-tag

FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence motif DYKDDDDK.[1] It has been used for studying proteins in living cells and for protein purification by affinity chromatography. It has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits, because its mild purification procedure tends not to disrupt such complexes. It has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by x-ray crystallography.

A FLAG-tag can be used in many different assays that require recognition by an antibody. If there is no antibody against a given protein, adding a FLAG-tag to a protein allows the protein to be studied with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence or detection by SDS PAGE protein electrophoresis and Western blotting.

The peptide sequence of the FLAG-tag from the N-terminus to the C-terminus is: DYKDDDDK (1012 Da). Additionally, it may be used in tandem, commonly the 3xFLAG peptide: DYKDHDG-DYKDHDI-DYKDDDDK (with the final tag encoding an enterokinase cleavage site). It can be fused to the C-terminus or the N-terminus of a protein, or inserted within a protein. Some commercially available antibodies (e.g., M1/4E11) recognize the epitope only when it is present at the N-terminus. However, other available antibodies (e.g., M2) are position-insensitive. The tyrosine residue in the FLAG-tag can be sulfated, which can affect antibody recognition of the FLAG epitope.[2] The FLAG-tag can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or myc-tag.

History

The first use of epitope tagging was described by Munro and Pelham in 1984.[3] The FLAG-tag was the second example of a fully functional, improved epitope tag, published in the scientific literature[4][5][6] and was the only epitope tag to be patented.[7][8] It has since become the most commonly used protein tag in laboratories worldwide. Unlike some other tags (e.g. myc, HA), where a monoclonal antibody was first isolated against an existing protein, then the epitope was characterized and used as a tag, the FLAG epitope was an idealized, artificial design, to which monoclonal antibodies were raised. The FLAG tag's structure was optimized for compatibility with proteins it is attached to, in that it is more hydrophilic than other common epitope tags and therefore less likely to denature or inactivate proteins to which it is appended. In addition, N-terminal FLAG tags can be removed readily from proteins once they have been isolated, by treatment with the specific protease, enterokinase (enteropeptidase).

The third report of epitope tagging, (HA-tag),[9] appeared about one year after the Flag system had been sent to laboratories throughout the world for beta-testing as a kit for recombinant protein production.

FLAG epitope tagging has grown to become an essential technology in most molecular biology laboratories around the world. In the decades since its original description, it has been applied to study virtually every disease and condition of mankind, and has been adopted in most fields of biology outside of medicine as well. Many companies that provide research tools and supplies offer the FLAG system, making it the first commercially successful nanotechnology device.

References

  1. ↑ Hopp, Thomas P.; Prickett, Kathryn S.; Price, Virginia L.; Libby, Randell T.; March, Carl J.; Pat Cerretti, Douglas; Urdal, David L.; Conlon, Paul J. (1988). "A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification". Bio/Technology. 6 (10): 1204–10. doi:10.1038/nbt1088-1204.
  2. ↑ Hunter MR, Grimsey LG, Glass M (2016). "Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model". Scientific Reports. 6 (27316). doi:10.1038/srep27316. PMC 4895180Freely accessible. PMID 27273047.
  3. ↑ Munro, S; Pelham, HR (1984). "Use of peptide tagging to detect proteins expressed from cloned genes: Deletion mapping functional domains of Drosophila hsp 70". The EMBO Journal. 3 (13): 3087–93. PMC 557822Freely accessible. PMID 6526011.
  4. ↑ Hopp, Thomas P.; Prickett, Kathryn S.; Price, Virginia L.; Libby, Randell T.; March, Carl J.; Pat Cerretti, Douglas; Urdal, David L.; Conlon, Paul J. (1988). "A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification". Bio/Technology. 6 (10): 1204–10. doi:10.1038/nbt1088-1204.
  5. ↑ Einhauer, A.; Jungbauer, A. (2001). "The FLAG™ peptide, a versatile fusion tag for the purification of recombinant proteins". Journal of Biochemical and Biophysical Methods. 49 (1–3): 455–65. doi:10.1016/S0165-022X(01)00213-5. PMID 11694294.
  6. ↑ http://www-users.med.cornell.edu/~jawagne/FLAG-tag.html[]
  7. ↑ FLAG is a registered trademark of Sigma-Aldrich Co. LLC
  8. ↑ Hopp, T.P., et al. (1987) Synthesis of protein with an identification peptide (vectors). United States Patent 4,703,004.
  9. ↑ Field, J; Nikawa, J; Broek, D; MacDonald, B; Rodgers, L; Wilson, IA; Lerner, RA; Wigler, M (1988). "Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method". Molecular and Cellular Biology. 8 (5): 2159–65. PMC 363397Freely accessible. PMID 2455217.
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