Biomedical Engineering Reference
In-Depth Information
Fig. 11.5
The principle of BRET-based assay.
a
To have an energy transfer between a donor
(light-producing enzyme) and an acceptor (fluorophore), the emission spectrum of the donor
must overlap with the excitation spectrum of the acceptor.
b
The energy transfer can occur only
when the donor and the acceptor are spatially close. BRET signal will be measured using fusion
with protein of interest when the two proteins studied physically interact. (Reprinted from Ref.
[
70
] for
a
and
b
with permission from John Wiley & Sons, Inc).
c
Odorant-induced BRET sig-
nal measurement. Crude membranes from three different clones co-expressing hOR1740-Rluc
and hOR1740-EGFP were used to perform BRET assays without or with odorants (helional as
a hOR1740 agonist)
d
BRET level variation upon hOR1740 stimulation with different helional
concentrations. (Reprinted from Ref. [
71
] for
c
and
d
with permission from ASBME)
in vivo
monitoring of biological events via signal cascades such as gene expression
and protein-protein interaction. Such technical progresses have led to the develop-
ment of sensitive and selective bio-analytical tools like recombinant whole-cell
biosensors. In order to investigate protein-protein interactions, one protein is fused
to the donor and the other to the acceptor. If the two fusion proteins interact and
the distance between the energy donor and acceptor is less than 10 nm, a resonance
energy transfer occurs and an additional light signal corresponding to the acceptor
reemission can be detected (see Fig.
11.5
).
Fluorescent methods which include FRET, utilize specific changes in confor-
mation or interaction of fluorescently labeled molecules as a result of a biological
process, to produce an increase or decrease in fluorescence [
72
]. Such interactions
can be measured within, or independently of a cell. German et al. described an
insect OR protein by the FRET technique [
73
]. Fusion proteins containing cyan
fluorescent protein (CFP) or yellow fluorescent protein (YFP) were produced us-
ing the baculovirus-mediated expression. In another example, the use of FRET-
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