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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.


Cleavage dependent activation of CP234-Luc/ENLYFQX fusion proteins. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. The X-axis is the amino acid at the P1' position (X) of the CP234-Luc/ENLYFQX fusion protein. The Y-axis is the fold activation of the CP234-Luc/ENLYFQX fusion proteins calculated by dividing the mean TEV digested values by the mean undigested values (no TEV digest), N = 3. Error bars are the standard deviation of the mean.
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Figure 3: Cleavage dependent activation of CP234-Luc/ENLYFQX fusion proteins. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. The X-axis is the amino acid at the P1' position (X) of the CP234-Luc/ENLYFQX fusion protein. The Y-axis is the fold activation of the CP234-Luc/ENLYFQX fusion proteins calculated by dividing the mean TEV digested values by the mean undigested values (no TEV digest), N = 3. Error bars are the standard deviation of the mean.

Mentions: To enable both a comprehensive and easy method for interrogating protease specificity, we created a vector which carries the CP234 -Luc coding region with two unique restriction sites inserted at the native luciferase N and C termini to facilitate the cloning in of protease recognition sequences. Using this vector, we generated twenty bioluminescent protease substrates: CP234-Luc/ENLYFQX proteins with all possible amino acids at the P1' position, to assess the utility this assay for examining protease substrate specificity. Nineteen of the twenty were activated to various degrees upon TEV protease digest as determined by both an increase in luminescence (Fig. 3) as well as the appearance of the smaller 34 kDa and 26 kDa CP234-Luc protein fragments (data not shown). The CP234-Luc/ENLYFQP protein was not activated by or digested by TEV protease. The fold activations of the proteins after protease digestion ranged from none (P1' = proline) to 420 (P1' = serine). Gel analysis of the digested luciferase fragment showed qualitatively similar results, although precise quantitation is difficult (data not shown). These results suggest that most amino acids are tolerated at the P1' position, with the exception of proline. This is consistent with previous reports [8].


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)

Cleavage dependent activation of CP234-Luc/ENLYFQX fusion proteins. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. The X-axis is the amino acid at the P1' position (X) of the CP234-Luc/ENLYFQX fusion protein. The Y-axis is the fold activation of the CP234-Luc/ENLYFQX fusion proteins calculated by dividing the mean TEV digested values by the mean undigested values (no TEV digest), N = 3. Error bars are the standard deviation of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Cleavage dependent activation of CP234-Luc/ENLYFQX fusion proteins. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. The X-axis is the amino acid at the P1' position (X) of the CP234-Luc/ENLYFQX fusion protein. The Y-axis is the fold activation of the CP234-Luc/ENLYFQX fusion proteins calculated by dividing the mean TEV digested values by the mean undigested values (no TEV digest), N = 3. Error bars are the standard deviation of the mean.
Mentions: To enable both a comprehensive and easy method for interrogating protease specificity, we created a vector which carries the CP234 -Luc coding region with two unique restriction sites inserted at the native luciferase N and C termini to facilitate the cloning in of protease recognition sequences. Using this vector, we generated twenty bioluminescent protease substrates: CP234-Luc/ENLYFQX proteins with all possible amino acids at the P1' position, to assess the utility this assay for examining protease substrate specificity. Nineteen of the twenty were activated to various degrees upon TEV protease digest as determined by both an increase in luminescence (Fig. 3) as well as the appearance of the smaller 34 kDa and 26 kDa CP234-Luc protein fragments (data not shown). The CP234-Luc/ENLYFQP protein was not activated by or digested by TEV protease. The fold activations of the proteins after protease digestion ranged from none (P1' = proline) to 420 (P1' = serine). Gel analysis of the digested luciferase fragment showed qualitatively similar results, although precise quantitation is difficult (data not shown). These results suggest that most amino acids are tolerated at the P1' position, with the exception of proline. This is consistent with previous reports [8].

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.