A polyhistidine-tag is an amino acid motif in proteins that consists of at
least six histidine residues, often at the N- or C-terminus of the protein. It
is also known as hexa histidine-tag, 6xHis-tag, His6 tag and by the
trademarked name His-tag. The tag was invented by Roche, although the use of
histidines and its vectors are distributed by Qiagen. Various
purification kits for histidine-tagged proteins are available from Qiagen,
Sigma, Thermo Scientific, GE Healthcare, Macherey-Nagel, Clontech, Bio-Rad, and
others. The use of the tag for academic users is
unrestricted; however, commercial users must pay royalties to Roche. The
original patent expired on 11 Feb 2003, and thus should be now public property;
current claims to royalties are based on a much narrower set of more recent
patents. Suitable tag sequences are available free for commercial use; for
example, MK(HQ)6 may be used for enhanced expression in E. coli and tag
removal. The total number of histidine residues may vary in the tag. The
his-tag may also be followed by a suitable amino acid sequence that
facilitates a removal of the polyhistidine-tag using endopeptidases.
This extra sequence is not necessary if exopeptidases are used to remove
N-terminal His-tags. Furthermore, exopeptidase cleavage may solve the
unspecific cleavage observed when using endoprotease-based tag removal.
Polyhistidine-tags are often used for affinity purification of genetically
modified proteins. Applications
= Protein purification = Polyhistidine-tags are often used for
affinity purification of polyhistidine-tagged recombinant
proteins expressed in Escherichia coli and other prokaryotic expression
systems. Bacterial cells are harvested via centrifugation and the resulting
cell pellet lysed either by physical means or by means of detergents and
enzymes such as lysozyme or any combination of these. At this stage raw
lysate contains the recombinant protein among many other proteins originating
from the bacterial host. This mixture is incubated with an affinity resin
containing bound bivalent nickel or cobalt ions, which are available
commercially in different varieties. Nickel and cobalt have similar
properties and as they are adjacent period 4 transition metals). These
resins are generally sepharose/agarose functionalised with a chelator, such as
iminodiacetic acid and nitrilotriacetic acid for nickel and
carboxylmethylaspartate for cobalt, which the polyhistidine-tag binds with
micromolar affinity. The resin is then washed with phosphate buffer to remove
proteins that do not specifically interact with the cobalt or nickel ion.
With Ni-based methods, washing efficiency can be improved by the
addition of 20 mM imidazole. Generally nickel-based resins have higher binding
capacity, while cobalt-based resins offer the highest purity. The purity and
amount of protein can be assessed by SDS-PAGE and Western blotting.
Affinity purification using a polyhistidine-tag usually results in
relatively pure protein when the recombinant protein is expressed in
prokaryotic organisms. Depending on downstream applications, including the
purification of protein complexes to study protein interactions, purification
from higher organisms such as yeasts or other eukaryotes may require a tandem
affinity purification using two tags to yield higher purity. Alternatively,
single-step purification using immobilized cobalt ions rather than
nickel ions generally yields a substantial increase in purity and
requires lower imidazole concentrations for elution of the his-tagged protein.
Polyhistidine-tagging is the option of choice for purifying recombinant
proteins in denaturing conditions because its mode of action is dependent
only on the primary structure of proteins. Generally for this sort of a
technique, histidine binding is titrated using pH instead of imidazole binding—at
a high pH, histidine binds to nickel or cobalt, but at low pH, histidine becomes
protonated and is competed off of the metal ion. Compare this to antibody
purification and GST purification, a prerequisite to which is the proper
folding of proteins involved. Polyhistidine-tag columns retain several
well known proteins as impurities. One of them is FKBP-type peptidyl prolyl
isomerase, which appears around 25kDa. Impurities are generally eliminated
using a secondary chromatographic technique, or by expressing the
recombinant protein in a SlyD-deficient E. coli strain. Alternatively
cobalt-based resins do not bind SlyD from E. coli and can be used for a
single-step purification [1]. = Separating One from Two Polyhistidine
Tags = Proteins with different numbers of
polyhistidine tags elute differently from nickel-affinity resin. For proteins
with a single hexahistidine tag, 75 mM imidazole enables elution from Ni-NTA,
whereas for proteins with two hexahistidine tags, 100 mM imidazole is
required for elution. This step-wise elution may be used to isolate specific
protein assemblies from a mixture, such as defined heteromultimers. Such an
approach was used in isolation of monovalent streptavidin.
= Binding assays = Polyhistidine-tagging can be used to
detect protein-protein interactions in the same way as a pull-down assay.
However, this technique is generally considered to be less sensitive, and
also restricted by some of the more finicky aspects of this technique. For
example, reducing conditions cannot be used, EDTA and many types of detergents
cannot be used. Recent advances in dual polarisation interferometry is amenable
to EDTA and a wider use of reagents, and the use of such site-specific tags
greatly simplifies the direct measurement of associated conformational
change. = Fluorescent Tags =
Hexahistadine CyDye tags have also been developed. These use Nickel covalent
coordination to EDTA groups attached to fluorophores in order to create dyes
that attach to the polyhistidine tag. This technique has been shown to be
effective for following protein migration and trafficking. There has
also been recent discoveries that show this technique may be effective in order
to measure distance via Fluorescent Resonance Energy Transfer.
Adding Polyhistidine Tags The most common polyhistidine tags are
formed of six histidine residues - which are added at the N-terminus preceded by
Methionine or C-terminus before a stop codon, in the coding sequence of the
protein of interest. The choice of the end where His-tag is added will depend
mainly on the characteristics of the protein and the methods chosen to remove
the tag. Some ends are buried inside the protein core and others are important
for the protein function or structure. In those cases the choice is limited to
the other end. On the other hand, most available exopeptidases can only remove
the His-tag from the N-terminus; removing the tag from the C-terminus
will require the use of other techniques.
There are two ways to add polyhistidines. The most simple is to
insert the DNA encoding the protein in a vector encoding a His-tag so that it
will be automatically attached to one of its ends. Another technique is to
perform a PCR with primers that have repetitive histidine codons right next
to the START or STOP codon in addition to several bases from one end of the DNA
encoding the protein to be tagged. Example of primer designed to add a
6xHis-tag using PCR. Eighteen bases coding six histidines are inserted right
after the START codon or right before the STOP codon. At least 16 bases
specific to the gene of interest are needed next to the His-tag. With 6 His,
the protein will have an added 1 kDa of molecular weight. Note: often, a linker
is placed between the protein of interest and the 6 His tag. This is to
prevent the polyhistidine tag from affecting the activity of the protein
being tagged. Detection
The polyhistidine-tag can also be used to detect the protein via
anti-polyhistidine-tag antibodies or alternatively by in-gel staining with
fluorescent probes bearing metal ions. This can be useful in subcellular
localization, ELISA, western blotting or other immuno-analytical methods.
Immobilization The polyhistidine-tag can be
successfully used for the immobilization of proteins on a surface such as on a
nickel- or cobalt-coated microtiter plate or on a protein array.
See also Protein tag
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