Hexahistidine tag

Six consecutive histidines. The imidazole side chains coordinate immobilized nickel or cobalt ions, so a 6xHis-tagged protein is captured on metal affinity resin and eluted with imidazole or low pH. The tag is small (0.84 kDa) and is detected by western blot.

Length: 18 bp (6 aa)

Uses: purification, epitope

Cleavable: No

Detected by: nickel affinity resin, cobalt affinity resin, anti-His antibody

Origin: Synthetic run of six histidines

Characteristics

Encodes HHHHHH (6 amino acids, 18 nucleotides; 0.84 kDa). The imidazole rings of consecutive histidines form coordination bonds with Ni2+, Co2+, Cu2+ or Zn2+ immobilized on a matrix, and 20-250 mM imidazole or low pH elutes the protein. Binding depends on tag length. With dihydrofolate reductase on Ni2+-NTA in 6 M guanidine HCl, His6 is retained at more than 90% and His3 at less than 10%, while His3 is retained at about 90% or more in phosphate buffer. The tag sits at either terminus; the better position is protein-specific.

Applications: Immobilized metal affinity purification of recombinant proteins from bacteria, yeast, mammalian cells and baculovirus-infected insect cells, and pull-down assays. It is the most common fusion partner in structural studies, used in about 60% of crystallographic studies. Tagged proteins are detected by western blot.

Limitations: It does not improve solubility. As an N-terminal fusion to 23 human proteins poorly expressed in E. coli KRX, 22% were soluble, against 52% for MBP and 39% for GST. E. coli proteins with affinity for divalent metals co-purify. Imidazole can interfere with NMR and crystallization, and metalloproteins are not recommended because NTA absorbs the metal.

Sequence

You must be signed in to view the full sequence.

References

  1. K. Terpe (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol - Terpe 2003 Tag Fusions
  2. Ohana et al. (2009). HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification. Protein Expr Purif - Ohana 2009 HaloTag7
  3. Bell et al. (2013). To fuse or not to fuse: what is your purpose?. Protein Sci - Bell 2013 Fusion Purpose