6xHis

Fig. 21 Chitin binding of 6xHis-tagged resilin with chitin-binding domain (6 H-resChBD) as compared to 6xHis-tagged resilin without chitin-binding domain (6 H-res). T total protein after affinity chromatography purification, B bound protein eluted from chitin beads, UB unbound protein. Reproduced from [187] with permission from The American Chemical Society, copyright 2009...

These columns are used for purification of recombinant fusion proteins tagged to 6XHis. The commercial columns contain the precharged Ni coupled to a tetradentate chelating absorbent such as the NTA (nitrilotriacetic acid), bound to a matrix which could be Sepharose or Cellulose. 

Fig. 4. Visualization of several purified RANTES proteins on a Coomassie stained SDS page Lanes 1 and 2, 6xHis-tagged RANTES expressed in the periplasmic space of E. coli Lane 3, Met-RANTES expressed in the cytoplasm of E. coli and purified from inclusion bodies Lane 4, eucaryotic expressed 6xHis-tagged RANTES.

Binding of 6xHis-tagged proteins to Ni-NTA agarose gel is pH dependent. The best binding is achieved at a pH higher than 7.5. 

In general, elution of 6xHis-tagged proteins does not take place at a pH higher than 4.5. However, elution conditions have to be optimized for every protein. If the protein is unstable at low pH, elution can also be performed with imidazole, a histidine analog. 

Crowe, J., Dobeli, H., Gentz, R., Hochuli, E., Stuber D., Henco, K., 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein ex-pression/purification. Methods Mol. Biol. 1994, 31, 371-387. 

Fig. 5. Circular dichroism spectroscopy of peptide (120-189) fused to a 6xHis-tag. The circular dichroism spectrum of 6xHis-rCla h6 (120-189) (protein concentration was 27 piMin salt-free water) was recorded on a Jasco spectropolarimeter (J-810) at 185-260nm. Computer-assisted analysis of the data showed that the peptide is folded with 11% helix, 35% (3-sheet, 27% loop and 26% random structural elements.

Fig. 29 A barcode of three different chemical functionalities formed in a silica capillary via spatially-selective polymerization. It consists of segments of poly(bis-SorbPC) doped with Rhodamine-capped DPPE (red), poly(bis-SorbPC) doped with NBD-capped DOPE (green), and DOPC doped with Ni2+-charged DOGS-NTA. After the lipid pattern was formed, 6xHis-tagged Cerulean, a blue fluorescent protein, was injected into the capillary and bound selectively to the immobilized Ni2+ (blue). The capillary inner diameter is 50 pm. Reprinted with permission from [96]. Copyright 2007, American Chemical Society...

This 35-kDa protein was confirmed as the vCKBP by pierforming the same assays with two recombinant Ws, W RPVA35 and W Lister A35 from which the fully functional gene had been inactivated by insertion of the LacZ gene. Neither of these viruses produced a secreted protein that bound to I-chemokine. Furthermore, when the W Lister 35-kDa ORF was expressed with a G-terminal 6xHis-tag in the baculovirus system, or as an Fc fusion protein from stably transfected mammalian cells, binding of the recombinant products to... 

Serum-Adsorption Experiments Protein-resistance measurements were carried out by following the same procedure as above to form a PLL-g-PEG/PEG-NTA monolayer on the waveguide (or after the standard assay experiments of binding 6xHis-tagged proteins), and then the surface was fully regenerated by exposing it to EDTA (200 mM). After a flat baseline was reached, the control human serum was injected into the OWLS flow cell. After 15 min of equilibration, the flow cell was rinsed with HEPES-2 buffer and allowed to equilibrate for 30 additional minutes. The difference to the first baseline corresponds to the amount of non-specific adsorption of proteins. 

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