Biology Reference
In-Depth Information
OTHER REAGENTS
Transfection reagent, for example, Lipofectamine
®
2000 (Invitrogen)
BRET coelenterazine substrate DeepBlueC (PerkinElmer)
Amersham ECL Western blotting detection reagent (GE Healthcare)
13.2
METHODS
13.2.1
Generation of expression constructs
Functional expression of TAS2R receptors in mammalian cell lines requires that the
receptors carry an additional sequence at their N-terminus (e.g., the 45 N-terminal
amino acids of the rat somatostatin receptor 3, “rsstr3”), which facilitates export
of the protein to the plasma membrane (
Bufe, Hofmann, Krautwurst, Raguse, &
Wolfgang Meyerhof, 2002
). TAS1Rs on the other hand do not need any N-terminal
modification to support their expression. For CoIP experiments, it is necessary to
further include a FLAG-tag at the C-terminus of the receptors, while BRET exper-
iments require constructs encoding for receptor-GFP
2
and receptor-
Renilla renifor-
mis
luciferase (Rluc) fusion proteins.
For easy and fast generation of the receptor constructs, we suggest to first build a
cloning cassette in a modular way (
Fig. 13.1
) in a mammalian expression vector (such
as pcDNA5/FRT). Then, the following approach can be followed: First, GFP
2
and Rluc
sequences are amplified from the BRET vector by PCR; the oligonucleotides should
additionally contain the sequences of the restriction enzymes 3 and 4, thereby adding
FIGURE 13.1
Modular cloning cassette for taste receptor expression constructs. A cloning cassette for
TAS2Rs is generated in a mammalian expression vector, for example, pcDNA5/FRT. Sites for
different restriction enzymes (RE) separate the modules from each other and enable easy
subcloning of the different parts. rsstr3: sequence of the first 45 amino acids of the rat
somatostatin receptor 3, used as N-terminal tag for TAS2Rs to enable functional plasma
membrane expression of the receptors; TAS2R: TAS2R coding sequence without stop codon;
FLAG: sequence of the FLAG-tag; GFP
2
: sequence of a modified green fluorescent protein
(GFP), used for BRET and CoIP experiments; Rluc: sequence of the luciferase of
Renilla
reniformis
, used for BRET experiments. TAS1R sequences can be cloned into the same
cassette using RE1 and RE3, replacing the combined sequences of rsstr3-TAS2R.
