Tag-lite® is a comprehensive selection of tools and reagents streamlined to specifically label fusion protein with synthetic HTRF dyes. Tag-lite combines HTRF with SNAP-tag®, CLIP-tag® and HaloTag® technologies. This solution is ideal for a wide range of applications, such as GPCR ligand binding assays and cell surface receptor mechanistic studies.
This platform is an original and efficient way to accurately label a protein of interest on a targeted site with HTRF dyes.
Originally developed by the Ecole Polytechnique Federale de Lausanne (EPFL) and commercialized by New England Biolabs, Inc., SNAP-tag and CLIP-tag are small fusion tags that covalently interact with dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker.
HaloTag, a Promega technology, is another orthogonal system for labeling proteins in living cells and in vitro. The HaloTag protein is a 33 kDa enzyme derivatized from a prokaryotic hydrolase.
As shown, SNAP-tag, CLIP-tag and HaloTag can easily be fused to either the N- or the C-terminal position on proteins of interest, and can then be specifically and covalently labeled with their defined substrates.
Cisbio Bioassays offers SNAP-tag, CLIP-tag, HaloTag encoding plasmids which are bordered by restriction sites for cloning a gene of interest.
A specific plasmid contruction can be engineered with the appropriate tag generic plasmid and the gene encoding for the protein of interest (e.g. GPCR). Once transfected into cells, this plasmid leads to the expression of the protein of interest fused to the SNAP-tag, CLIP-tag or HaloTag motif. Cisbio also proposes a list of validated plasmids encoding for GPCRs N-terminally fused with SNAP-tag, CLIP-tag or HaloTag.
A selection of three substrates is available for each SNAP-tag, CLIP-tag and HaloTag. These substrates are labeled with HTRF fluorophores: terbium cryptate donor (Lumi4-Tb), green or red HTRF acceptors.
SNAP-tag, CLIP-tag or HaloTag are suicide enzymes that react in a very specific way with their respective substrates. These enzymes metabolize their substrate and transfer part of this dye-coupled substrate to themselves. At the end of this irreversible complete process, the enzyme is no longer active, and the remaining tag becomes labeled with the dye. The fused protein labeled either to a donor or to an acceptor is ready to be combined with the other dye partner, and to get the new HTRF assay up and running.
Studies confirmed that SNAP-tag fusion on the GPCR N-terminal and its specific labeling with the appropriate fluorescent substrate do not affect either the GPCR binding of a specific ligand or the GPCR function. Vasopressin EC50s measured with IP1 accumulation assay were very similar on both WT-V1a and ST-V1a.
Reverse transfection of vasopressin 1a receptor was performed with the wild type (WT-V1a) and the SNAP-tag V1a (ST-V1a) of the receptor. Inositol 1-phosphate production induced by vasopressin stimulation was assessed using the IP-One kit on the two different cellular models.