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140 Charlton and Zachariou
and non-denaturing environments, it has been used to refold proteins whilst
still bound to the IMCC (14). This approach can allow the user to obtain
near homogenous, soluble protein from insoluble input material. Conversely,
incorporation of a histidine tag has been shown to improve the soluble yield
of some recombinant proteins by its presence alone, presumably by increasing
the hydrophilicity of the protein and thus rendering it more compatible with
expression in Escherichia coli (15).
As a mature affinity chromatographic technology, IMAC has seen application
in circumstances outside of its traditional role of protein purification. Significant
interest has been in proteomic screening technologies; with chelators immobilized
on magnetic beads, IMAC binding is amenable to automation. This allows
for rapid expression and purification of large protein libraries (16). IMAC has
also functioned as a coupling technique for immobilization of receptors in
microarrays (17) and as a tether for membrane proteins in the generation of
artificial lipid bilayers (18). Histidine tags have even been incorporated into
synthetic oligonucleotides, allowing for their purification by IMAC (19).
The ubiquitous application of histidine-tag IMAC has seen a range of
supporting technologies emerge; tools for the specific detection and removal
of histidine tags are commercially available. Qiagen’s TAGzyme system is a
classic example of the latter. The system consists of a series of three enzymes
that are specifically tailored to remove N-terminal hexahistidine tags leaving
no vector or tag-derived amino acids on the target protein. The system is
described in detail elsewhere in this book. Specific detection of histidine-tagged
proteins is as readily available as tag-bearing cloning vectors, with detection
systems supplied by Pierce, Novagen, Clontech, Qiagen and Invitrogen, among
many others. These systems usually rely on variations of an anti-hexahistidine
antibody for secondary antibody–reporter enzyme conjugate detection, or a
reporter enzyme (horseradish peroxidase and alkaline phosphatase) linked to
a chelator for direct metal chelate complex detection. Samples can then be
queried by either method for the presence of the histidine tag in a western blot
type assay format.
With a wealth of background literature, a wide variety of cloning vectors
and stationary supports, IMAC is a popular first choice for many recombinant
protein purification applications at any scale, from proteomic screening up to
biopharmaceutical production.
2. Materials
2.1. Purification of His-Tagged Proteins Using Ni-NTA
1. Stationary Support: Ni-NTA-Superflow (Qiagen).
2. Charge solution: 0.1 M NiNO 3 .