Polyhistidine-tag
A polyhistidine tag ( His-tag interchangeably, hexahistidine tag, His6 tag ) is a protein tag that is used for protein purification and detection of labeled proteins.
Properties
The amino acid sequence of the poly-histidine tag is a sequence of at least six histidines, the gene sequence is cloned N-terminally by the start methionine codon or C-terminally in front of the stop codon in the open reading frame of a gene. This creates a fusion protein with a polyhistidine tag. Occasionally an interface for a protease or an intein between the protein and poly-histidine tag is inserted, to allow cleavage of the tag after protein purification.
The polyhistidine tag binds with micromolar affinity for divalent nickel or cobalt ions to form a chelate. The ions are immobilized by binding to a solid -linked chelator proteins with poly-histidine tag can be used in affinity chromatography (more precisely, in a metal - chelate affinity chromatography ) can be selectively bound and removed by rinsing the column with 20 mM imidazole from the unbound proteins be freed. Column bed as is usually nitrilotriacetic acid agarose ( Ni-NTA agarose), less frequently used Ni2 agarose - iminodiacetic acid (Ni -IDA - agarose) or Co2 Carboxymethylaspartate agarose ( Co - CMA - agarose). The elution of bound proteins is carried out with a buffer containing 75-300 mM imidazole, an acidic pH ( pH 4 to pH 6, nickel - cobalt and agaroses - Agarose ), or nickel or cobalt ions. Interfering substances are EDTA and reducing agents, some surfactants (e.g., from the sample buffer). After elution, the imidazole is mostly removed by dialysis.
At the nickel -based column materials also binds the peptidyl- prolyl cis -trans isomerase FKBP - type ( SlyD, 25 kDa ) from E. coli. Therefore, often a tandem affinity purification is used for further purification. There are also SlyD -deficient bacterial strains. Cobalt - CMA - agarose binds SlyD weaker.
Through a serial use of multiple polyhistidine tags proteins can be eluted successively with increasing number with increasing imidazole concentration. Characterized several proteins can be simultaneously purified with a different number of hexahistidine and eluted with increasing concentrations of imidazole in succession.
Applications
Polyhistidine tags for purification of recombinant proteins by affinity chromatography, for pull-down assays and - used for methods with an immune marker ( eg, ELISA, Western blot, immunofluorescence, immunohistochemistry, and flow cytometry) - when using anti - polyhistidine tag antibody. There are selective binding fluorescent dyes. A protein with poly-histidine tag can be immobilized on nickel or cobalt are adsorbed at surfaces or nickel-containing membranes.