How many kda is flag tag

Protein complexes and protein-protein interactions constitute the functional bases of the life activities within the cell. Many tag combinations have been developed since the tandem affinity purification TAP technique appeared at late s [ 43 ]. TAP-tagging, which employs two sequential affinity purification steps, can significantly reduce the chance of contaminants retained in the eluate.

Double-affinity tag is an efficient approach for the purification protein complexes under native conditions [ 44 , 45 ]. As a powerful tool to separate interacting protein complex, TAP-tagging strategy is widely used in the studies of protein interaction networks. The combination of TAP technique with mass spectrometry MS has been widely adopted as a highly efficient method to identify and characterize the components of protein complexes [ 46 — 49 ].

Fortunately, many TAP expression vectors are commercially available today. Altogether, the adoption of tag in TAP strategy needs to carefully determine according to the advantages and disadvantages of various tags and the characteristics of a target protein.

To choose an effective combination, it is normally necessary to consider the abilities of the tags to improve the yield, enhance the solubility, and facilitate the purification of their fusion partners. Additionally, if affinity tags have the potential to interfere with structural or functional studies, the fused tag must be removed from the bait protein as follows.

The use of affinity tag for the purification of proteins in both prokaryotic and eukaryotic expression systems is a well-accepted method. In theory, it cannot be excluded that affinity tags, especially those with large size, may have the potential to interfere with the structure and function of the proteins.

If this circumstance happens, measures should be made for removing them. Any affinity tag, whether small or large, can be easily removed by introducing a specific protease recognition sequence between the tag and target protein Figure 1 b. The most frequently used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus TEV , and human rhinovirus 3C protease.

The advantages and disadvantages of these endopeptidases were thoroughly discussed in the previous works of the literature [ 1 , 52 — 54 ]. Moreover, other endopeptidases e. Enterokinase may sometimes cleave at other basic residues, depending on the conformation of the protein substrate [ 56 — 60 ].

In addition, the cleavage efficiency of enterokinase is closely associated with the amino acid residue after downstream of the recognition site [ 61 , 62 ]. It will not cleave at the recognition site if the recognition sequence is followed by proline. As the enterokinase, both thrombin and factor Xa are trypsin-like serine proteases which will cleave peptide bonds on the carboxyl side of a basic amino acid residue.

Similar to the enterokinase, factor Xa will not cleave at a site followed by proline or arginine. Thus, unlike enterokinase and factor Xa, thrombin cleavage may result in the retention of one or two amino-terminal amino acid residues in protein of interest. The TEV protease is a highly site-specific cysteine endoprotease. The cleavage activity of the enzyme can be stimulated by calcium ions [ 70 ], and an additional affinity step is needed for on column tag removal such as using immobilized sortase A [ 71 ].

The recombinant enzyme is specifically designed to facilitate removal of the protease by allowing simultaneous protease immobilization and cleavage of GST affinity tag. Besides the tag cleavage methods described above, removal of the tag without using a protease is also feasible by introducing a protein element with self-splicing capacity into a variety of tag-based purification systems.

These elements are naturally self-splicing proteins called inteins that can excise themselves from the parent protein [ 74 , 75 ]. Inteins can be designed at N- or C-terminal splice junctions to obtain self-cleaving inteins, which can then be used to achieve self-cleaving of various affinity tags.

The specific applications have been comprehensively reviewed by other authors [ 76 — 78 ]. Affinity purification is an approach of isolating biomoleculars from cell extracts based on a highly specific interaction as that between antigens and antibodies, as well as receptors and ligands. Affinity tag is absolutely necessary for many applications in life sciences, including the purification of protein of interest.

Although each tag has its specific advantages and disadvantages in purification efficiency, the versatile affinity purification systems with different affinity tags are powerful to isolate recombinant protein and protein complexes. To sum up, there is no universal affinity purification system for exploring different bait protein and its binding partners at present.

So far, it is feasible to generate multiple purified human proteins or protein domains for affinity proteomics and large-scale structural genomics studies by now [ 79 ].

National Center for Biotechnology Information , U. J Anal Methods Chem. Published online Dec Author information Article notes Copyright and License information Disclaimer. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC. Abstract Affinity tags have become powerful tools from basic biological research to structural and functional proteomics.

Introduction The expression and purification of recombinant proteins have become increasingly common for characterizing structure and function of proteins in recent years. Table 1 Common widely used affinity tags for purification of recombinant proteins. Open in a separate window.

Figure 1. Combinatorial Tagging Strategy and the Studies of Protein Interacting Partners Protein complexes and protein-protein interactions constitute the functional bases of the life activities within the cell. Removal of Affinity Tags The use of affinity tag for the purification of proteins in both prokaryotic and eukaryotic expression systems is a well-accepted method. Conclusions Affinity purification is an approach of isolating biomoleculars from cell extracts based on a highly specific interaction as that between antigens and antibodies, as well as receptors and ligands.

References 1. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expression and Purification. Waugh DS. Making the most of affinity tags. Trends in Biotechnology. Walls D, Loughran ST. Tagging recombinant proteins to enhance solubility and aid purification.

Methods in Molecular Biology. Crystal structures of fusion proteins with large-affinity tags. Protein Science. Comparison of affinity tags for protein purification. Prokaryotic expression, purification of a new tumor-relative protein FAM92A and its characterization in renal cell carcinoma. Cancer Letters. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline. Acta Crystallographica F. Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers. FEBS Letters. Sachdev D, Chirgwin JM. Fusions to maltose-binding protein: control of folding and solubility in protein purification.

Methods in Enzymology. Lebendiker M, Danieli T. Purification of proteins fused to maltose-binding protein. Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp.

Journal of Clinical Microbiology. Guo F, Zhu G. Presence and removal of a contaminating NADH oxidation activity in recombinant maltose-binding protein fusion proteins expressed in Escherichia coli.

Malhotra A. Tagging for protein expression. SUMO fusion technology for enhanced protein expression and purification in prokaryotes and eukaryotes.

Heterologous Gene Expression in E. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein.

Biochemical Journal. Avoid repeated freeze. Lead Time Basically, we can dispatch the products out in working days after receiving your orders. Delivery time maybe differs from different purchasing way or location, please kindly consult your local distributors for specific delivery time.

Review: Cells were lysed and immunoprecipitated using monoclonal IgG2b anti-Flag antibody. By Anonymous. Tel: Email: [email protected] Distributors Worldwide. Your Good Partner in Biology Research. View All pathways. Protocols References Download Center. How does Flag-tag work? Structure of Flag-tag 4. Development and discovery of Flag-tag 5. Properties 6. How to Flag-tag a protein? Size of Flag-tag and 3x Flag-tag 8. Specifications of the Flag-tag and 3x Flag-tag 9.

Flag-tag epitope tag sequences Applications Flag- vs. Myc-tag HA-tag Spot-tag V5-tag Flag-tag plasmids What is Flag-tag?