Polyhistidine

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. 

R. Lindeman and G. Zundel, Proton transfer in and polarizability of hydrogen bonds coupled with conformational changes in proteins. II. IR investigation of polyhistidine with various carboxylic acids, Biopolymers 17, 1285-1301 (1978). 

To conclude, an issue that is bringing great attention in recent years, the production of chimeric virus-hke particles should be briefly analysed. These chimeric VLPs are potentially valid systems for broader vaccine production, i.e. against a large number of different serotypes [34] in addition they can result in safe combination vaccines between closely related viruses [35], can be able to carry multiple foreign epitopes [36-39], or even, with the incorporation of tags (e.g. polyhistidine), allow easy single-step product recovery [40,41]. 

Alternatively, a terminal tag such as polyhistidine can be added to the target protein to improve the efficiency of the purification procedure [24]. An affinity column designed to recognize polyhistidine could be used to isolate the tagged protein.The polyhistidine is then cleaved and the mixture dialyzed. While this approach is useful for small-scale preparations, whether it can be used for large pharmaceutical scale preparations remains to be seen. 

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... 

Biophysical characterization showed that a single HRP II protein bound 17 molecules of heme [35]. In an in vitro heme polymerization assay, HRP II promoted the synthesis of hemozoin, while controls, such as the proteins bovine serum albumin and lysozyme or the homopeptides polyhistidine, polylysine, and polyasparagine, did not. FT-IR analysis of the reaction product showed the characteristic vibrations of hemozoin. The polymerization activity had a pH maximum near 4.0, which dropped off precipitously near the pKa of histidine. The heme polymerization... 

Fig. (3). Hemozoin Production Mediated by Bionucleating Templates. Representative polymerization assay with 50 pM of hemin in 2 ml acetate buffer (500 mM, pH 4.8) at 37° C. BNTI and BNTII were used in 1 and 2 nmol amounts. Chloroquine (CQ, 100 pM) was included with BNT I and BNT n in inhibition reactions. Polyhistidine and bovine serum albumin in approximately 1 and 2 nmol amounts were used in protein control experiments. The blank control was the acetate buffer above. Base line amounts of insoluble aggregate are consistent with those previously reported under similar conditions.

Polyhistidine is insoluble in water at pH 7.8 but is soluble at pH 5.5. Explain this observation. Would you expect the polymer to be soluble at pH 10 ... 

Wang, C.-Y. and Huang, L. (1984) Polyhistidine mediates an acid-dependent fusion of negatively charged liposomes. Biochemistry, 23, 4409 1416. 

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. 

O Brien SM, Sloane RP, Thomas OR, Dunnill P, Characterization of non porous magnetic chelator supports and their use to recover polyhistidine-tailed T4 lysozyme from a crude E. coli extract, I. Biotechnol., 54 53-67, 1997. 

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. 

We do not intend to consider basic poly(a-amino acid)s, such as polylysine or polyhistidine, among the main subjects of this review, since they have been the object of many other reviews, along with other poly(a-amino acid)s. 

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