FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a peptide protein tag that can be added to a protein using

recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fo ...

technology Technology is the application of Conceptual model, conceptual knowledge to achieve practical goals, especially in a reproducible way. The word ''technology'' can also mean the products resulting from such efforts, including both tangible too ...

, having the sequence DYKDDDDK (where D=

aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protei ...

, Y=

tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a conditionally essential amino acid with a polar side group. The word "tyrosine" is ...

, and K=

lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. Lysine contains an α-amino group (which is in the protonated form when the lysine is dissolved in water at physiological pH), an α-carboxylic acid group ( ...

). It is one of the most specific tags and it is an artificial antigen to which specific, high affinity monoclonal antibodies have been developed and hence can be used for protein purification by

affinity chromatography Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...

and also can be used for locating proteins within living cells. FLAG-tag has been used to separate recombinant, overexpressed protein from

wild-type The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, " ...

protein expressed by the host organism. FLAG-tag can also be used in the isolation of

protein complexes A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multidomain enzymes, in which multiple catalytic domains are found in a single polypeptide chain. Protein c ...

with multiple subunits, because FLAG-tag's mild purification procedure tends not to disrupt such complexes. FLAG-tag-based purification has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by

x-ray crystallography X-ray crystallography is the experimental science of determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract in specific directions. By measuring th ...

. A FLAG-tag can be used in many different assays that require recognition by an

antibody An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as pathogenic bacteria, bacteria and viruses, includin ...

. 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-tag sequence. Examples are cellular localization studies by

immunofluorescence Immunofluorescence (IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of anti ...

immunoprecipitation Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sam ...

or detection by SDS PAGE protein electrophoresis and

Western blotting The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detec ...

. The peptide sequence of the FLAG-tag from the

N-terminus The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...

to the C-terminus is: DYKDDDDK (1012 Da). Additionally, FLAG-tags may be used in tandem, commonly the 3xFLAG peptide: DYKDHD-G-DYKDHD-I-DYKDDDDK (with the final tag encoding an enterokinase cleavage site). FLAG-tag 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 An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although e ...

only when FLAG-tag 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 Sulfation (sometimes spelled sulphation in British English) is the chemical reaction that entails the addition of SO3 group. In principle, many sulfations would involve reactions of sulfur trioxide (SO3). In practice, most sulfations are effected ...

when expressed on certain secreted proteins, which can affect antibody recognition of the FLAG epitope. The FLAG-tag can be used in conjunction with other affinity tags, for example a polyhistidine tag (

His-tag A polyhistidine-tag, best known by the trademarked name His-tag, is an amino acid motif in proteins that typically consists of at least six histidine (''His'') residues, often at the N- or C-terminus of the protein. It is also known as a hexa hi ...

), HA-tag or myc-tag.

History

The first use of

tagging was described by Munro and Pelham in 1984. The FLAG-tag was the second example of a fully functional, improved epitope tag, published in the scientific literature. and was the only epitope tag to be patented. It has since become one of the most commonly used protein tags 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 sequence was optimized for compatibility with proteins it is attached to, in that FLAG-tag is more hydrophilic than other common epitope tags and therefore less likely to reduce the activity of proteins to which FLAG-tag 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 Enteropeptidase (also called enterokinase) is an enzyme produced by cells of the duodenum and is involved in digestion in humans and other animals. Enteropeptidase converts trypsinogen (a zymogen) into its active form trypsin, resulting in the ...

). The third report of epitope tagging, ( HA-tag), appeared about one year after the Flag system had been first shipped.

References

{{Protein tag Biochemical separation processes Biochemistry detection methods Laboratory techniques Molecular biology Peptide sequences Octapeptides

History

See also

References