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Detection of Protein–Protein and Protein–DNA Interactions 193
As mentioned above, the HaloTag system consists of chemically modified
HaloTag ligands which bestow different functionalities onto HaloTag fusion
proteins upon binding. To achieve efficient and specific binding with several
different ligands, we designed the ligands so that they consist of two elements:
the constant reactive group and a variable functional reporter group. The
reactive group consists of the chloroalkane, which is the natural substrate for
HaloTag protein. This part of the ligand is the same in all the ligands and is
involved in the covalent and specific binding to the HaloTag polypeptide. The
remaining part of the ligand, the functional group, encompasses many different
entities including different fluorescent dyes, affinity handles (e.g., biotin) or
the solid support (e.g. resin). Thus, binding of different HaloTag ligands to
HaloTag fusion protein imparts different functionalities onto the fusion protein
that allow imaging and/or immobilization. Consequently, one genetic construct
can be used in various in vitro and in vivo (cell-based) assays (see Fig. 1).
Immobilization of proteins onto solid support surfaces is becoming
increasingly important in characterization of protein function and protein interactions
(9). We have developed a surface for immobilization of HaloTag
fusion proteins, a nonmagnetic resin (HaloLink), which enables covalent
and oriented surface immobilization. HaloLink resin consists of agarose
Protein immobilization
Surface (HaloLink TM )
Biotin
Protein labeling
O
H
N
N
H
S
O
H N
O
O
O
O
O
O
Functional/
Reporter
group
O O
H N
O
O
l
C
Reactive
group
l
C
HaloTag
TMR
Ligand
N-terminus
TMR
diAcFAM
Coumarin
HaloTag ligands
+
N
2 - O H
N
O
C
O
O
N
H
N
O
O
O
O
O
O
O
2
H N
O
O
O
O
O
O
H N
O
l
O
C
O
l
C
Cl
HaloTag protein
C-terminus
Fig. 1. Variable functionalities of the HaloTag Technology. The HaloTag
Technology comprises the HaloTag protein and a system of interchangeable synthetic
ligands that specifically and covalently bind to the HaloTag protein. These ligands
bind to HaloTag impart multiple functions to a HaloTag fusion protein including
imaging and immobilization. Thus, one genetic construct can be used in various in
vitro and in vivo assays.