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Promega @cademy webinar:
"Bioluminescent assays to monitor protease activity"

 

The Promega @cademy provides a tailored programme of non-commercial live webinars, covering a wide range of scientific topics, from very basic concepts to highly technical research presentations. This free webinar was held on Monday June 6, 2011. The title is: "Bioluminescent assays to monitor protease activity". To view the webinar recordings, please click on the link below.
 
 

Specialist WebinarsThis is a Specialist webinar, these webinars addresses more specialized topics and are useful for PhD students and senior scientists.
 
 

Information

Proteases are important targets for drug discovery because of their involvement in myriad cell signaling pathways. Recent FDA approval for protease inhibitors of DPPIV, the proteasome, and renin as drugs for type II diabetes, multiple myeloma, and hypertension, respectively, attest to their importance. Rapid and sensitive assays of proteolytic activity are necessary for the general characterization of proteases and high-throughput screening for protease inhibitors. Fluorescence has been widely used for monitoring protease activity, but fluorogenic substrates can exhibit high background and many compound libraries have a significant percentage of interfering fluorescent compounds. We have developed an alternative bioluminescent platform for protease assays. The bioluminescent chemistry based on firefly luciferase is particularly versatile. We have used the luciferin component in the reaction as a scaffold to create luminogenic substrates for a wide variety of proteases. By combining peptide-conjugated aminoluciferin substrates and a stabilized luciferase in a coupled-enzyme format, we have developed homogeneous protease assays that have low background, improved assay sensitivity, increased dynamic range, and greater flexibility in read time.

 Examples comparing luminescent protease assays to fluorescent protease assays will be shown and the differences in kinetics will be explained. Both cell-based assays and biochemical assays using purified proteases have been developed; the specific requirements and utility for each will be discussed. We have optimized luminescent assays for several protease targets, including caspases, DPPIV, calpain, the proteasome, and deubiquitinating enzymes, but have capabilities for a much wider array of targets. Using a coupled-enzyme system for high throughput screening has unique considerations that will be discussed. In addition, we have recently combined luminescent substrates to create a general protease assay designed for detecting contaminating proteases.

In addition to using luciferin as a scaffold, we have engineered luciferase to be a flexible vector that can be used to create a substrate for a protease of interest. This novel technology complements the aminoluciferin-based technology and allows rapid generation of protease substrates through molecular cloning and coupled transcription/translation cell-free expression, thus enabling the facile evaluation of protease function. Our bioluminescent platform for protease assays utilizes both luciferin and engineered luciferase as scaffolds for generating substrates; the combined technologies can provide solutions for a wide variety of protease assay needs.

 


This webinar is be presented by Martha O'Brien Ph.D., Sr. Scientist at Research - Biochemical & Cellular Technologies, Promega Corporation.


 

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