16 research outputs found
A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening
Exploring the TemperaturePressure Phase Diagram of Staphylococcal Nuclease
The temperature dependence of the pressure-induced equilibrium unfolding of staphylococcal nuclease (Snase) was determined by fluorescence of the single tryptophan residue, FTIR absorption for the amide I‘ and tyrosine O−H bands, and small-angle X-ray scattering (SAXS). The results from these three techniques were similar, although the stability as measured by fluorescence was slightly lower than that measured by FTIR and SAXS. The resulting phase diagram exhibits the well-known curvature for heat and cold denaturation of proteins, due to the large decrease in heat capacity upon folding. The volume change for unfolding became less negative with increasing temperatures, consistent with a larger thermal expansivity for the unfolded state than for the folded state. Fluorescence-detected pressure-jump kinetics measurements revealed that the curvature in the phase diagram is due primarily to the rate constant for folding, indicating a loss in heat capacity for the transition state relative to the unfolded state. The similar temperature dependence of the equilibrium and activation volume changes for folding indicates that the thermal expansivities of the folded and transition states are similar. This, along with the fact that the activation volume for folding is positive over the temperature range examined, the nonlinear dependence of the folding rate constant upon temperature implicates significant dehydration in the rate-limiting step for folding of Snase
Determination of the Volume Changes for Pressure-Induced Transitions of Apomyoglobin between the Native, Molten Globule, and Unfolded States
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ∼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 °C or in culture medium for
>15 h at 37 °C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ∼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 °C or in culture medium for
>15 h at 37 °C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes
Transgenic reporter mice expressing Caspase 3/7 GloSensor.
<p>A) Schematic of the pCLEX Caspase 3/7 GloSensor transgene construct. B) Excision of the floxed EGFP-stop cassette when crossed with a Cre expressing mouse strain should result in tissue specific transcription of the reporter. C) Representative bioluminescence images of bi-transgenic (for the reporter and p48-Cre) or mono-transgenic (transgenic for the reporter in the absence of Cre) animals pre- and 30 hrs post-cerulein injection (75 ug/kg, total of 12 injections in 48 hrs). D) Quantification of BLI signal induction upon cerulein treatment. E) Representative bioluminescent and fluorescent (EGFP) ex-vivo images of pancreata from mono- or bi-transgenic animals. F) Representative bioluminescence images of bi-transgenic (right) or mono-transgic (left) animals.</p
Assessing drug sensitivity of rare and transient cell populations.
<p>A) FACS analysis of dissociated D54 cells sorted into CD133<sup>+</sup> and CD133<sup>−</sup> populations, P3 represents the CD133 expressing cell population. B) to E) Bioluminescence assay of CD133<sup>+</sup> and CD133<sup>−</sup> sorted D54 cells incubated with 200 ng/ml TRAIL (B), 50 µM MNS (C), 50 µM MK886 (D) or 12.5 µM GW7647 (E). Bioluminescence was plotted as fold induction over values obtained from vehicle treated cells. Experiments were performed in triplicates and plotted as mean ± SEM. Paired t-test was performed for all experiments and * denotes p<0.05 value at indicated time points.</p