Histidine tag

Fig. 15 Amino acid sequences of artificial extracellular matrix (aECM) proteins. Each protein contains a TV tag, a histidine tag, a cleavage site, and elastin-like domains with lysine residues for crosslinking. The RGD cell-binding domain is found in aECM 1, whereas aECM 3 contains the CS5 cell-binding domain. aECM 2 and aECM 4 are the negative controls with scrambled binding domains for aECM 1 and aECM 3, respectively. Reprinted from [121] with permission from American Chemical Society, copyright 2004...

Dorn, 1. T., Neumaier, K. R. and Tampe, R. (1998) Molecular recognition of histidine-tagged molecules by metal-chelating lipids monitored by fluorescence energy transfer and correlation spectroscopy. ]. Am. Chem. Soc 120, 2753. 

Wegner GJ, Lee NJ, Marriott G, Com RM (2003) Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions. Anal Chem 75 4740-4746... 

Kato K, Sato H, Iwata H (2005) Immobilization of histidine-tagged recombinant proteins onto micropattemed surfaces for cell-based functional assays. Langmuir 21 7071-7075... 

Fancy, D.A., Melcher, K., Johnston, S.A., and Kodadek, T. (1996) New chemistry for the study of multiprotein complexes The six-histidine tag as a receptor for a protein crosslinking reagent. Cbem. Biol. 3, 551-559. 

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

Bricker, T. M, J. Morvant, N. Masri, H. M. Sutton, L. K. Frankel (1998) Isolation of a highly active photosystem n preparation from Synechocystis 6803 using a histidine tagged mutant of CP 47. Biochim. Biophys. Acta, 1409 50-57... 

We have also used mutagenic primers to insert histidine tags and epitope tags into cDNAs of interest (Neish et al, 2003). Inserting several codons into a cDNA is a little more difficult than simply substituting one amino acid for another, but is quite feasible using this technique. Optimization of the reaction is absolutely critical when performing more difficult manipulations such as this. 

Janknecht, R., G.de Martynoff, J.Lou, R. A.Hipskind, A.Norandeim and H.G.Stunnenberg. (1991) Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia vims. Proc Natl Acad Sci USA, 88, 8972-8976. 

Among these amino acids histidine is the most commonly used one. Attachment of histidine tags to the recombinant proteins polypeptides is the most known development in the field of IMAC. Histidine and other metal affinity tags are widely used for protein purification [26], Adsorbents may be prepared by binding chelators onto the surface and metals to the chelators. Free coordination sites of the metal ions are needed for the analyte to bind to metal ions [25]. 

Crofcheck, C., Loiselle, M Weekley, J Maiti, 1., Pattanaik, S., Bummer, PM., and Jay, M. (2003). Histidine tagged protein recovery from tobacco extract by foam fractionation. Biotechnol. Prog. 19(2) 680-682. 

Strugnell, S.A., B.A. Wiefling, and H.R Deluca, A modified pGEX vector with a C-terminal histidine tag recombinant double-tagged protein obtained in greater yield and purity. Anal Biochem, 1997. 254(1) 147-9. 

A variety of useful protein tags are available. A common one is a histidine tag, often just a string of six His residues. A poly-His sequence binds quite tightly to metals such as nickel. If a protein is cloned so that its sequence is contiguous with a His tag, it will have the extra His residues at its carboxyl terminus. The protein can then be purified by chromatography on columns with immobilized nickel. These procedures are convenient but require caution, because the additional amino acid residues in an epitope or His tag can affect protein activity. 

The complex was produced in E. coli cells from the cloned genes allowing for some "engineering" of the proteins. A ten-histidine "tag" was added at the N termini of the P subunits so that the complex could be "glued" to a microscope coverslip coated with a nickel complex with a high affinity for the His tags. The y subunit shafts protrude upward as shown in Fig. 18-16. The y subunit was mutated to replace its... 

Metal affinity columns can be used for the purification of antibodies with a hexa-Histidine tag. Immobilized metal affinity chromatography is incompatible, in our own experience, with direct loading of antibodies in supernatants... 

Expression vector with suitable promotor, multiple cloning site, and fusion tag, where applicable (e.g., six-histidine tag). 

Key Words Ligation-independent cloning T7 expression system pET vector hexa-histidine tag. 

Protease inhibitor cocktail (for histidine-tagged proteins) (Sigma-Aldrich, St. Louis, MO). 

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