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A novel bioluminescent protease assay using engineered firefly luciferase.

Wigdal SS, Anderson JL, Vidugiris GJ, Shultz J, Wood KV, Fan F - Curr Chem Genomics (2008)

Bottom Line: Understanding their biological functions underpins the efforts of drug discovery.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.

View Article: PubMed Central - PubMed

Affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.

ABSTRACT
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.

No MeSH data available.


TEV protease dose dependent activation of CP234-Luc/ENLYFQS protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes with titrating amounts of TEV protease and luminescence was measured from 5 µL aliquots in 100 µL 1:1 diluted Bright GloTM Luciferase Assay reagent in dH2O; N = 4. Results are plotted as signal to noise (S/N). The limit of detection was defined as the amount of TEV protease giving a S/N ratio = 3 (dotted line). The R2 value was 0.99.
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Figure 4: TEV protease dose dependent activation of CP234-Luc/ENLYFQS protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes with titrating amounts of TEV protease and luminescence was measured from 5 µL aliquots in 100 µL 1:1 diluted Bright GloTM Luciferase Assay reagent in dH2O; N = 4. Results are plotted as signal to noise (S/N). The limit of detection was defined as the amount of TEV protease giving a S/N ratio = 3 (dotted line). The R2 value was 0.99.

Mentions: To determine the sensitivity and linear range of the CP234-Luc/ENLYFQS assay, a TEV enzyme titration was performed. The luminescence from the CP234-Luc/ENL-YFQS assay was dependent on the TEV enzyme concentration. The signal to noise (S/N) was calculated from the luminescent values and is defined as the mean signal – mean background divided by the standard deviation of the background (Fig. 4). The limit of detection was defined as the amount of TEV protease detected at a S/N of 3 (i.e., 3 standard deviations higher than the background). Thus the limit of detection was 0.163 mU TEV protease per 100 μL Bright GloTM Luciferase Assay sample. The linear range of the assay was ≥ 1,000 fold (0.163 to 167 mU (the highest concentration tested in this experiment) of TEV protease per 100 μL Bright GloTM Luciferase Assay sample).


A novel bioluminescent protease assay using engineered firefly luciferase.

Wigdal SS, Anderson JL, Vidugiris GJ, Shultz J, Wood KV, Fan F - Curr Chem Genomics (2008)

TEV protease dose dependent activation of CP234-Luc/ENLYFQS protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes with titrating amounts of TEV protease and luminescence was measured from 5 µL aliquots in 100 µL 1:1 diluted Bright GloTM Luciferase Assay reagent in dH2O; N = 4. Results are plotted as signal to noise (S/N). The limit of detection was defined as the amount of TEV protease giving a S/N ratio = 3 (dotted line). The R2 value was 0.99.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803436&req=5

Figure 4: TEV protease dose dependent activation of CP234-Luc/ENLYFQS protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes with titrating amounts of TEV protease and luminescence was measured from 5 µL aliquots in 100 µL 1:1 diluted Bright GloTM Luciferase Assay reagent in dH2O; N = 4. Results are plotted as signal to noise (S/N). The limit of detection was defined as the amount of TEV protease giving a S/N ratio = 3 (dotted line). The R2 value was 0.99.
Mentions: To determine the sensitivity and linear range of the CP234-Luc/ENLYFQS assay, a TEV enzyme titration was performed. The luminescence from the CP234-Luc/ENL-YFQS assay was dependent on the TEV enzyme concentration. The signal to noise (S/N) was calculated from the luminescent values and is defined as the mean signal – mean background divided by the standard deviation of the background (Fig. 4). The limit of detection was defined as the amount of TEV protease detected at a S/N of 3 (i.e., 3 standard deviations higher than the background). Thus the limit of detection was 0.163 mU TEV protease per 100 μL Bright GloTM Luciferase Assay sample. The linear range of the assay was ≥ 1,000 fold (0.163 to 167 mU (the highest concentration tested in this experiment) of TEV protease per 100 μL Bright GloTM Luciferase Assay sample).

Bottom Line: Understanding their biological functions underpins the efforts of drug discovery.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.

View Article: PubMed Central - PubMed

Affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.

ABSTRACT
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.

No MeSH data available.