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Promega notes 100: novel biosensors to monitor cellular events in live cells

LIVE-CELL BIOSENSOR Novel Biosensors to Monitor Cellular Events in Live Cells Review of Fan, F. et al. (2008) Novel genetically encoded biosensors using firefly luciferase. ACS Chem. Biol. 3, 346–51.
Neal Cosby, Promega Corporation entists targeted the hinge region of the luciferase mol- Drug discovery and life science researchers desire to ecule using three design strategies, covalent, nonco- collect meaningful data that advance scientific valent and allosteric, to modulate enzyme activity knowledge. To accomplish this, often the data need to be gathered in a biologically relevant context. As To create the cAMP biosensor, for example, the of cellular events such the technologies available to scientists have scientists constructed a circularly permuted luciferase advanced, the demand for cell-based assays, espe- by placing new N- and C-termini in the middle of GPCRs can be obtained cially live-cell assays, has increased.
the protein. They then connected the native N- in minutes following Recently Promega scientists published the first and C-termini with a cAMP binding moiety of pro- treatment using thistechnology.
peer-reviewed article on a new technology that incor- tein kinase A (RIIβB). The resulting mutant form porates genetically modified forms of firefly luciferase of luciferase emits increasing amounts of light in used to detect molecular events within living cells response to increasing concentrations of cAMP.
(1). The paper demonstrates multiple approaches toconstructing biosensors using circularly permuted ornonpermuted forms of luciferases. In brief, the sci- Figure 1. Design strategies for biosensors using firefly luciferase. A. Molecular models of firefly luciferase in the absence of substrates (open
conformation) or bound to an analog of the luciferyl-adenylate reaction intermediate (closed conformation, analog colored yellow) generated using
PDB files 1LCI and 2D1S, respectively.The smaller C-terminal domain of luciferase (residues 441–544) is predicted to rotate and translocate toward
the larger N-terminal domain during the catalytic cycle. Residues 4–233 and 234–544 are colored blue and gray, respectively, for the ‘open' conforma-
tion. Residues 4–355 and 359–544 are colored blue and gray, respectively, for the ‘closed' conformation. Nonvisible residues at the N- and C-termini
of PDB file 1LCI were typically excluded from the various biosensor design strategies. B. Schematic representation of the three design strategies
used to generate luciferase biosensors. Covalent. Fusion of the wild-type N- and C-termini with a polypeptide containing a protease cleavage site
inhibits formation of the closed conformation. Cleavage by protease relieves this constraint, allowing increased luminescence. Noncovalent.
Association of polypeptides FRB and FKBP12 in the presence of rapamycin inhibits formation of the closed conformation, causing decreased
luminescence. Allosteric.The conformational change of an analyte binding domain modulates luminescence, e.g., cAMP binding to RIIβB promotes
increased luminescence. Reprinted with permission from Fan, F. et al. (2008) ACS Chem. Biol. 3, 346–51.
PROMEGA NOTES WWW.PROMEGA.COM NUMBER 100 SEPTEMBER 2008 LIVE-CELL BIOSENSOR is required. A standard luminometer with injectors is suf- Intracellular biosensor technologies remain an area of keen ficient to detect signal readout and obtain real-time interest that until recently was dominated mostly by flu- kinetic data, all without any loss in the information that orescent approaches. The most common examples are can be collected. As evident in Figure 2, signal kinetics FRET-based biosensors using variants of GFP. However, of reversible agonists and antagonists can be obtained in FRET-based biosensors suffer from low dynamic range of minutes following treatment. Important to HTS applica- response due to characteristics inherent in the detection tions, these assays can be performed at room temperature modality (2). The results are FRET ratios of 30–100% or with little difference in the range of response and more 2-fold response at best, which can be a significant draw- stable kinetics.
back. In contrast, the genetically encoded firefly luciferasebiosensor described by Fan et al. (1) exhibits 20-fold and greater response. This response is 10 times that of FRET.
Louis Hodgson wrote in his review of the biosensor In addition, the detection of cAMP-mediated cellular article in ACS Chemical Biology, "Making these events by the luciferase biosensor is reversible. Figure 2 genetically encoded chemiluminescence sensors is no illustrates the large change in light output and revers- easy task"(2). The GloSensor™ cAMP Assay(a), developed ibility of the luminescent signal when the biosensor is using Promega's expertise in bioluminescence, greatly used to interrogate endogenous β2-adrenergic receptor simplifies this task for researchers. We encourage those activity in HEK293 cells.
interested in better understanding this new tech- The key advantages of genetically encoded luciferase nology to view the original paper for a detailed descrip- biosensors are better dynamic range and assay simplicity.
tion of how the various biosensors were developed and Following stable or transient transfection of the biosen- how they function. sor, the cells are pre-equilibrated with the substrate andthen treated with compounds that are known or suspect- ed to modulate intracellular cAMP. There are no addi- 1. Fan, F. et al. (2008) ACS Chem. Biol. 3, 346–51.
tional steps, and no specialized instrumentation or software 2. Hodgson, L. (2008) ACS Chem. Biol. 3, 335–7.
To learn more about the new GloSensor™ cAMP
Assay product offerings please visit:
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(a)For research use only by not-for-profit institutions subject to the purchaser's prior agreement to the terms of the limited use label license. Use by for-profit institutions,use for commercial purposes such as for payment or other consideration, and use inanimals each requires additional licensing. This product and/or its use is subject to Figure 2. Allosteric cAMP biosensor. Signal kinetics and reversibility
one or more of the following Promega patent applications: U.S. Pat. Appln. Ser. Nos.
in living cells at 37 ºC. HEK293 cells transiently expressing CP359- 09/645,706, 10/943,508, 10/957,433, 11/316,042, 11/732,105, 11/786,785, 11/825,304, PCT Pat. Appln. Ser. No. PCT/US2007/008176 and various corresponding patent appli- βB were treated with 10 mM isoproterenol (ISO) or 10 mM cations and issued patents.
forskolin (FSK) alone. Modulated cells were treated sequentially with10 μM ISO, 10 μM propranolol (PRO), and 10 μM FSK (n = 3).
GloSensor is a trademark of Promega Corporation.
Reprinted with permission from Fan, F. et al. (2008) ACS Chem. Biol. 3,
346–51.
NUMBER 100 SEPTEMBER 2008

Source: http://promega.in/~/media/files/resources/promega%20notes/100/novel%20biosensors%20to%20monitor%20cellular%20events%20in%20live%20cells.pdf?la=en