6 His tag; 6 His epitope tag; Hexa His tag; HHHHHH epitope tag; HHHHHH tag; His tag; Polyhistidine Tag
Citations
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Background
It is the His-tag – one of the most common protein purification and detection labels. Insert it at the N- or C-terminus of the target protein and affinity chromatographers purify the protein. His-tag is also detectable by antigen-antibody immune responses. It's not that the histidine tag's size matters for the protein's shape or function.
His-tag is a short sequence of 6 to 10 consecutive histidine residues, most common His tag with six histidine residues. The tag can be placed at the N- or C-terminus of the target protein. It is most important for the purification of recombinant proteins through IMAC. The histidine in the His-tag could coordinate with divalent metal ions (like Ni2+ or Co2+) embedded in the resin, which allowed scientists to purify the target protein from large cell lysates. The purification process usually involves eluting His-tagged proteins using imidazole. Aside from purification, the His-tag is used for protein identification. You can identify the target protein by anti-His-tag antibodies, commonly by Western blotting and ELISA. Also, His-tag can be used in experiments with protein-protein interactions and protein localisation.
The advantages of the His-tag lie in its compactness and simplicity. Its small molecular weight typically does not affect the structure and function of the target protein. Also, it enables ultra-pure target proteins to be purified via one-step IMAC. The His-tag can be used with bacteria, yeast, plants and mammals. Also, His-tag is flexible: purification can occur under native or denaturing conditions. Compared to other tags, the His-tag can be purified using inexpensive and readily available nickel or cobalt resins, eliminating the need for proprietary and costly alternatives. Furthermore, it has low immunogenicity, which usually means that the tag does not need to be removed during antibody production. Lastly, the positioning of the His-tag is more flexible, providing researchers with more options for experimental design.
Figure 1. Schematic representation of basic cloning strategies and commonly used purification modalities (Source: Wood DW. 2014)
Alternative Names
Anti-6xHistidine Tag monoclonal antibody
References
1. Wood DW. New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Bio. 2014 Jun;26:54-61.
2. Mishra V. Affinity Tags for Protein Purification. Curr Protein Pept Sci. 2020;21(8):821-830.
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References
Angiotensin II induces the exocytosis of galectin-3 via integrin alpha v/AKT/NF-kappa B signaling pathway
EUROPEAN REVIEW FOR MEDICAL AND PHARMACOLOGICAL SCIENCES
OBJECTIVE: To explore the role of integrin alpha v in Angiotensin II (Ang II)-induced exocytosis and endocytosis of galectin-3 (gal-3) in vascular smooth muscle cells (VSMCs). MATERIALS AND METHODS: A primary culture of mouse VSMCs was established by the enzymatic digestion of aorta. Adeno-Cre was used to specifically knockdown integrin alpha v. VSMCs were treated with Ang II, LY294002 (inhibitor of AKT signaling pathway), and Bay11-7082 (inhibitor of nuclear factor-kappa B, NF-kappa B), respectively. Endocytosis of His-tagged gal-3 was analyzed by immunofluorescence. The Western blot was performed to detect the protein level in cell supernatant and lysate. RESULTS: Ang II increased the exocytosis of gal-3 and activated AKT and NF-kappa B signaling pathways. The knockdown integrin alpha v effectively decreased the activation of AKT and NF-kappa B signals and the exocytosis of gal-3 induced by Ang II, but it had a little effect on the endocytosis of gal-3. Ang II increased the phosphorylation of AKT and NF-kappa B through integrin alpha v. AKT is the upstream signal of the NF-kappa B signaling pathway. LY294002 or Bay11-7082 could decrease Ang II-induced exocytosis of gal-3 in VSMCs. CONCLUSIONS: Ang II, depending on integrin alpha v/AKT/NF-kappa B signaling pathway, induced the exocytosis of gal-3.
Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag
PROTEIN EXPRESSION AND PURIFICATION
Authors: Abd Elhameed, Heba A. H.; Hajdu, Balint; Balogh, Ria K.; Hermann, Eniko; Hunyadi-Gulyas, Eva; Gyurcsik, Bela
The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3'-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.
COA
Publication 
Figure 1. Schematic representation of basic cloning strategies and commonly used purification modalities
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