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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.
 This
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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