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192 Urh et al.
and affinity purification tags. Autofluorescent proteins revolutionized the way
protein function is studied in living cells (1–3). They are useful not only for
protein localization studies but also for study of dynamic processes, conformational
changes and protein–protein interactions. Similarly, affinity fusion tags
transformed in vitro analysis of proteins. Affinity tags provide a selective, easy
and efficient tool for protein isolation and immobilization (4–7). The number of
new affinity tags and applications for their use continues to grow (8). However,
there are limitations to both technologies. With autofluorescent proteins, we
are limited with respect to fluorophores, and in addition, these proteins do
not provide us with an easy option to isolate and immobilize proteins for in
vitro studies. The use of an additional tag, for example, His tag, is required
for protein immobilization when using fluorescent proteins. On the other hand,
affinity tags provide a very efficient method for in vitro protein studies, but
they do not enable specific labeling and imaging of proteins in live cells.
Our goal was to develop a new technology that will combine advantages
of both of these technologies and overcome some of the limitations. Based
on these criteria, we have developed the HaloTag technology that enables
specific labeling, imaging and immobilization of proteins in vivo and in vitro.
The technology is a based on a new protein fusion tag, called HaloTag, and
a series of synthetic HaloTag ligands which specifically and covalently bind
the HaloTag protein.
HaloTag is a monomeric protein of 33 kDa and can be genetically fused
to the protein of interest either at the C or N terminus using a HaloTag
expression vector. The HaloTag protein was derived from a hydrolase found
in Rhodococcus rhodochrous, and therefore, it is not present in mammalian
systems, insect cells, yeast and even Escherichia coli. Thus, HaloTag
technology does not suffer from the interference of an endogenous protein
or ligand, which enhances the specificity of this system. The first and most
important modification of the wild-type enzyme was introduction of a mutation
that leads to preservation of the covalent bond and a permanent association of
the protein with the substrate. We used the natural substrate to develop a series
of chemically modified HaloTag ligands (see Fig. 1).
In addition to the critical modification in the active site which leads to
covalent binding of the ligand, other mutations were introduced into the binding
pocket. These mutations dramatically increase the rate of binding between
HaloTag protein and the HaloTag ligands. Fluorescence polarization
analysis using fluorescent HaloTag ligand and purified GST-HaloTag
fusion protein shows that the binding kinetics of the ligand to HaloTag protein
is very rapid with an on-rate similar to that measured for the biotin–streptavidin
interactions.