From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 193

The necessary tool kit to design a new FRET-based biosensor
(kinase activity reporter)

Substrate

Docking
site

Linker(s)

Fp#1
Donor

Development of a new
FRET-based biosensor
(“KAR”)

Phospho-
amino-Acid
binding
domain

Fp#2
Acceptor

Targeting
sequnece

Screening in
bacteria colonies

Screening in
mammalian cells

FRET efficiency measurements

If necessary... to optimize FRET efficiency

Figure 5.22 Overall methodology for creation and/or optimization of KAR biosensors.

In the light of opportunities offered by newly engineered FPs, many
studies suggest alternatives to conventional FRET pairs. Combining the
YFP or GFP as a donor with orange or red fluorescent proteins as acceptor
is now possible. Although these FRET pairs have mostly been used in pro-
tein interactions studies, they have recently emerged as an alternative to the
CFP/YFP FRET pair in biosensor design. 42
Figure 5.22 illustrates the process necessary for the creation and/or the
optimization of KAR biosensors.

6.5. Targeting FRET biosensors to subcellular compartments
Biosensors can be directed at distinct subcellular compartments in order to
gain information on the compartmentalized activity of a kinase of interest.
This is achieved by adding an additional amino acid sequence at the
N-terminal or C-terminal extremity of the sensor. Several subcellular targeting
sequences have been isolated and are now well characterized ( Table 5.3 ).

6.6. In vitro and in cellulo characterization of new FRET
biosensors
Two paths can be exploited to test newly built and optimized biosensors:
in vitro and in cellulo . Note that results from one approach may not necessarily
be similar to those generated with the other one. However, characterization