Polyhistidine "tag

Nickel affinity chromatography was chosen as the primary purification technique because it is a fast and reliable one-step assay and purified complexes can often be used in downstream applications without the necessity of removing the polyhistidine tag. In addition, the polyhistidine tag is smaller than many other affinity tags targeted by commercially available affinity resins and, in most cases, does not seem to interfere with the structure and function of the recombinant protein. 

PS1 The PS 1-prep, introduced in this communication is the first reported with a polyhistidine tag fused to the N-terminus of the PsaF subunit. This construct was possible due to the fact that cyanobacterial PsaF-deletion mutants show no impact on photoautotrophic growth - in contrast to Chlamydomonas reinhardtii, where inactivation of PsaF results in a severe reduction of electron transfer from plastocyanin to PS 1 [Hippier et al. 1997], Also, the N-terminus of the F-subunit which was decorated by the tag is located towards the lumen side which enables an attachment of the isolated PS1 with the lumen-exposed /donor-side to the electrode surface in our hydrogen-producing device. 

Another technique is to engineer genes to place a polyhistidine "tag" at the C terminus of a protein chain. Commercial cloning vehicles and kits are available for this purpose.124 The protein produced when the engineered gene is expressed can be captured by the affinity of the polyhistidine tag for Cu2+, Ni2+, Co2+, or Zn2+ held in chelated form on an affinity column.124-127... 

Fig. 4. The classic rotation experiment (A) Fj (asj6sy) is immobilized on a NiNTA modified glass surface via polyhistidine tags introduced into the N-termini of the [1-subunits. A fluorescently labeled actin filament is attached to the y-subunit via biotin/...

Most biocatalytic conversions are performed with the enzyme immobilized in the microreactor. Miyazaki et al. [426] developed a simple noncovalent immobilization method for His-tagged enzymes on a microchannel surface. These enzymes contain a polyhistidine-tag motif that consists of at least six histidine residues, often located at the N- or C-terminus. The H is-tag has a strong affinity for nickel and can be reversibly immobilized by a nickel-nitrilotriacetic acid (Ni-NTA) complex (Scheme 4.103), a strategy commonly used in affinity chromatography. 

This mode of separation, as the name suggests, uses stationary phases with a special affinity for a specific analyte. The affinity ligand immobilized on the stationary phase varies dramatically from peptide, to protein, to oligonucleotide, to monoclonal antibody. In some cases the target molecule is labelled with an affinity tag to simplify the separation. This approach is common in the synthesis of recombinant proteins where the system can be engineered so that the target biomolecule expresses a tag such as polyhistidine. A stationary phase functionalized with aminodiacetic acid and nickel chelate is then used to fish out the required molecule by chelating with the polyhistidine tag. 

Nuclear receptor LBDs were engineered to have an amino-terminal polyhistidine fusion tag. Constructs were expressed in E. coli strain BL21 (DE3) using the T7 promoter. PPARa LBD was also expressed as a polyhistidine tagged recombinant in baculovirus infected T. ni. 

In parallel, various affinity systems were also investigated such as the specific interactions between maltose-binding protein and positively charged polypyrrole film or a metal affinity immobilization concept based on the coordination of polyhistidine-tagged proteins or DNA to metal centers such as Ni " or chelated by polypyrrole films containing carboxylate, imidazole, or nitrilotriacetic acid groups (NTA) [15]. 

The fabrication of DNA sensors was thus carried out by the electropolymerization of the chelating pyrrole-NTA ligand followed by the successive coordination of Cu " and ssDNA modified by a polyhistidine tag [16]. Then, the labeless impedimetric detection of a ssDNA from the human immunodeficiency virus was performed via its hybridization onto the polypyrrole surface. The resulting detection limit (10 mol L ) was markedly better than those currently recorded for impedimetric DNA sensors. 

Another well established immobilization technique is via recombinant tags like polyhistidine tags. The idea came from immobilized metal affinity chromatography (IMAC) for the purification of proteins tagged with polyhistidine [145]. This method allows immobilization without prepurification steps and shows no interference with the enzyme structure or function, and does not affect the secretion or folding of fusion proteins within cells [146]. 

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