Addition of an epitope to a protein coding sequence: FLAG-tag

Some technical problems for the adequate analysis of the expression of cloned cDNA are the lack of functional assays and/or specific antibodies (Ab) to the proteins produced. To overcome this difficulty fusion proteins where a known peptide is fused to the expression product have been described. In addition to the Flag-Tag, His-tag and GST-tag are both widely used.

The flag peptide that was first used was a 11-amino-acid leader peptide of the gene-10 product from bacteriophage T7 fused at the amino-terminus of GAL4 (yeast transcription factor). At the time, there were no anti-GAL4 Ab commercially available (1), so a fusion protein with an epitope recognized by a commercially available antibody was prepared. The most widely used hydrophilic octapeptide now is DYKDDDDK (2) though recent studies suggest that a shorter peptide, DYKD, can be recognized with almost the same affinity by the M1 monoclonal antibody (3). Also, new tag sequences have been described for other monoclonal antibodies. The peptide MDFKDDDDK is recognized by M5 and MDYKAFDNL recognized by M2 (4). The binding reaction is also dependent on Calcium, so proteins can frequently be eluted from an affinity matrix by EDTA containing buffer (16). The beauty of this system is that it allows for the tag to be placed at either the amino-terminus (5) carboxy-terminus (6), or in association with other tags (7). It will not usually interfere with the fusion protein expression, proteolytic maturation or activity (8, 17). So far, even if the tag is placed in the MHC class I molecule, it will not interfere with either alloantibody recognition or cytotoxic T cell-MHC interactions (9).

The system provides immediate advantages such as purifying a fusion protein by anti-Flag affinity chromatography (2), or in one-step (3) and two-step (10) procedures and being able to use most of the immunological techniques that involve monoclonal antibody (ELISA, Western blot, FACS, immunohistochemistry, etc.). The most recent uses of this technique are in transfection of eucaryotic cells (11), mammalian cells (12), insect cells (6) and even transgenic systems (13). Even more exciting is the idea that this system may help solve one of the mayor problems in adenovirus gene transfer by targeting the cells with the use of bispecific antibodies. These bispecific antibodies bind to the flag peptide as well as to some specific receptor on the cell surface, for example the alpha v integrin in Wickham's experiment (14).

A mayor disadvantage of this system has been recently described. This tag system depends on specific detection of flag fusion proteins and no cross-reactivity to cellular proteins. Schafer et al.(15) have just isolated a rat cDNA clone coding for a new isoform of a beta phosphatase by screening a rat brain expression library with monoclonal antibody M2. It seems that five out of the eight amino acid residues in a sequence motif were identical. Even though this is a problem to consider, the FlAG-tag system is a powerful tool that has to be evaluated whenever Ab are not available to detect a fusion protein.

1. Witzgall R; O'Leary E; Bonventre JV. A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail. Anal Biochem, 1994 , 223:2, 291-8

2. Chubet RG, Brizzard BL. Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells. Biotechniques 1996;20(1):136-141

3. Knappik A, Pluckthun A. An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments. Biotechniques 1994;17(4):754-761

4.Slootstra JW, Kuperus D, Pluckthun A, Meloen RH. Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5. Mol Divers 1997;2(3):156-164

5. Guimarães MJ; Peterson D; Vicari A; Cocks BG; Copeland NG; Gilbert DJ; Jenkins NA; Ferrick DA; Kastelein RA; Bazan JF; Zlotnik; A. Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism? Proc Natl Acad Sci U S A, 1996, 93:26, 15086-91

6. Mazumdar M; Mikami A; Gee MA; Vallee RB In vitro motility from recombinant dynein heavy chain. Proc Natl Acad Sci U S A, 1996, 93:13, 6552-6

7. Robeva AS; Woodard R; Luthin DR; Taylor HE; Linden. Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure. J Biochem Pharmacol, 1996, 51:4, 545-55

8. Molloy SS; Thomas L; VanSlyke JK; Stenberg PE; Thomas G. Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J, 1994, 13:1, 18-33

9.Fulton JE; Thacker EL; Bacon LD; Hunt HD. Functional analysis of avian class I (BFIV) glycoproteins by epitope tagging and mutagenesis in vitro. Eur J Immunol, 1995, 25:7, 2069-76

10. Herren B; Pech M . Expression of a rat PDGF receptor beta ectodomain generates a low affinity ligand antagonist. J Recept Res, 1993, 13:1-4, 725-38

11. Hunt N; Ruppert J; Willey K; Schulte HM. Epitope mapping of the human TSH receptor; structure function studies. Exp Clin Endocrinol Diabetes, 1996, 104 Suppl 4:, 52-5

12. Chubet RG; Brizzard BL. Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells. Biotechniques, 1996, 20:1, 136-41

13. Oster-Granite ML; McPhie DL; Greenan J; Neve RL. Age-dependent neuronal and synaptic degeneration in mice transgenic for the C terminus of the amyloid precursor protein. J Neurosci, 1996, 16:21, 6732-41

14 Wickham TJ; Segal DM; Roelvink PW; Carrion ME; Lizonova A; Lee GM; Kovesdi I. Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies. J Virol, 1996, 70:10, 6831-8

15. Schäfer K; Braun T. Monoclonal anti-FLAG antibodies react with a new isoform of rat Mg2+ dependent protein phosphatase beta. Biochem Biophys Res Commun, 1995, 207:2, 708-14

16. Prickett, Amberg, and Hopp (1989) Biotechniques 7: 580-589.

17. Knappik and Pluckthun (1994) Biotechniques 7: 754-61.