Hexahistidine-tagged protein

Kamoto M, Umezawa N, Kato N, Higuchi T (2009) Turn-on fluorescent probe with visible light excitation for labelling of hexahistidine tagged protein. Bioorg Med Chem Lett 19 2285-2288... 

High-Throughput Purification of Hexahistidine-Tagged Proteins Expressed in E. coli... 

Purification of the proteins from the cultures grown in 24-well blocks can be performed in 96-well filter plates using a vacuum manifold for column forming, washing, and elution steps, as outlined below. The hexahistidine-tagged proteins in cleared cell lysates are batch loaded onto Ni-NTA agarose in a 96-well filter plate. Columns are formed by applying low (200 mbar) vacuum pressure. These mini-columns are extensively washed, and subsequently the proteins are eluted into a microtiter plate. 

Baek, S. H., et al. (2004), Surface plasmon resonance imaging analysis of hexahistidine-tagged protein on the gold thin film coated with a calix crown derivative, Biotechnol. Bioprocess. Eng., 9,143-146. 

Kimple AJ, Muller BE, Siderovski DP et al (2010) A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance. Methods Mol Biol 627 91-100... 

Thompson LB, Mack NH (2010) Bifunctional polyacrylamide based polymers for the specific binding of hexahistidine tagged proteins on gold surfaces. Phys Chem Chem Phys 12 4301 308... 

Fig. 1. Various schematics of bead display for molecular assemblies on beads. The Py subunits of the G protein (circles labeled with [i and y) are fused with either FLAG or hexahistidine tag, which recognizes the biotinylated M2 anti-FLAG antibodies on streptavidin-coated beads or chelated nickel on the dextran-treated beads. A socket and plug connecter is utilized to depict the very high-affinity interaction of the epitope tag. This modular setup allows for either a subunit (for capturing FPR-GFP) or as subunit (for capturing / 2AR-GFP) to be coupled with the fly subunit to form the complete G protein coating the bead. Fluorescent components such as GFP or ligand are indicated in green. See text for details.

Key Words Protein expression screen hexahistidine tag dot blot. 

The E. coli expression system used is based on the pQE-80L expression system. Sequences inserted into the multiple cloning site can be expressed as native proteins bearing an N-terminal hexahistidine tag upon IPTG induction of the T5 promoter in suitably transformed E. coli cells. 

Figure 1.1 Affinity purification scheme. RNA X denotes the desired target RNA to be purified 6x His indicates the hexahistidine tag that is added to the MS2/MBP fusion protein to yield protein HMM. Figure is adapted from Batey and Kieft (2007).

The introduction of hexahistidine tag (Hise-tag) to the N-terminus of chimeric protein significantly simplify the protein purification procedure, enabling isolation of target protein directly from crude cell extract (Efremenko et al, 2006). There were two main tasks in this work was i) to obtain the genetic construct encoding synthesis of fusion protein N-Hise-X-OPH, where X = superecliptic-pH-sensitive fluorine ii) to reveal conditions (host-strain, temperature and inductor concentration) favorable for construct expression in E.coli cells. 

Synthesis of the diabody was carried out by means of a modification of the method reported in Refs [4.17, 4.18] for synthesis of the ior-CEAI murine antibody scFv fragment. A non-covalent dimer association was obtained from two identical ior-CEAI scFv antibody fragments, in which VL and Vl domains were linked via a 5 amino acid residue. A hexahistidine tag at the C-terminus of the Vl domain aided protein purification. 

Figure 2 Western blot obtained from a SDS PAGE ofE. coli proteins 4 hours after medium shift. Amino acids added as indicated at the top of the figure. DHFR was detected with antibodies raised against the amino terminal hexahistidine tag...

Both hexahistidine-tagged and native forms of the recombinant E. coli enzyme have been purified. " The activity of the native enzyme was assessed by monitoring its ability to catalyze transfer of the [1- " C] octanoyl group from [l- " C]octanoyl-ACP to apo-H-protein. The protein exhibited a turnover number of 0.2 s, and values for octanoyl-ACP and apo-H-protein of 10.2 4.4pmoll and 13.2 2.9p,moll respectively. 

The first evidence of in vitro turnover was reported in a study from the Gronan and Marietta laboratories using both native LipA and a form containing a G-terminal hexahistidine tag. " Purification of the hexahis-tidine-tagged protein from the soluble fraction of crude lysate was performed under anaerobic conditions, and analysis of the resulting as-isolated protein, which was dark brown in color, revealed the presence of 3.4 0.4 atoms of iron and 4.8 0.8 atoms of sulfide per monomer. Subsequent EPR analysis of the dithionite-reduced protein again showed the presence of [4Fe S] clusters, although spin quantification indicated less than 5% of the reduced form. 

Kapanidis, AN, Ebright, YW, and Ebright,RH, Site-specific incorporation of fluorescent probes into protein Hexahistidine-tag-mediated fluorescent labeling with (Ni2+ nitrilotriacetic acid)(N)-fluorochrome conjugates. Journal of the American Chemical Society 123 (2001) 12123-12125. 

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