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A breakthrough technology for protease assay
Fluorescence Based Protease Assay Cleaves its Natural Protein Substrate
Technology
Despite that the natural substrates of proteases in living cells are full length proteins, short peptides labeled by a fluorescence dye and a quencher are widely used as substrates in proteases activity assays. This assay is quantitative and convenient. However, since a short linear peptide does not have the specific conformation of its parent protein, the assay suffers two serious drawbacks: First, the overall conformation of the substrate protein can be critical to the reaction specificity. Apparently, these interactions do not exist for a peptide substrate. Second, inhibitors potentially bind to the substrate protein cannot be identified. In addition, the pH dependent and other reaction conditions of a peptide substrate can be very different from those of the natural protein substrate.
To solve these problems, we developed a novel protease assay that cleaves its natural protein substrate. In the assay, the substrate is fluorescently labeled in a site specific manner by protein ligation method. The reaction progress of protease cleavage was monitored as a continuous kinetic curve without need to stop the reaction. This is assay is as convenient and quantitative as peptide substrate assay. So far, we have successfully developed and implemented the assay to the cleavage reactions of
  • Adam 17 or Adam 10 to pro-TNF alpha protein
  • Adam 17 or Adam 10 to pro-TGF alpha protein
  • Beta-secretase to Amyloid Precursor Protein
  • Renin to Angiotensinogen
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        Example 1
        Cleavage of pro-TNF-alpha by Adam 17
            Example 2
            Cleavage of APP by BACE
We provide custom development of this assay to other protease. Please email us for a quote.
Eliminate the Artifacts of Short Peptide Substrates
In addition to the lack of conformation, a short peptide substrate suffers another serious problem: the fluorescence group and its quenching pair are often labeled at the positions very close to the cleavage site (2-4 residues away). These two groups are very large compare to the most amino acid side chains. They can introduce serious artificial interactions between the substrate and its protease. Because the FERT based fluorescence quench is heavily distance dependent (quench efficiency is proportional to the reversed 6th power of the distance between the fluorescence and its quenching groups), it is not possible to place the two groups away from the cleavage site. As the results, the kinetic parameters, pH dependence, inhibitor profiles and other properties of the reaction are often seriously departed from its true values.
In our native protein substrates, the single fluorescence is far away from cleavage site (15 or more residues). This eliminates the artificial caused by the labeled group. The figure shown below is an example to show the importance to use the native protein substrate. With our pro-TNF-alpha protein substrate, the optimal pH of Amad17 cleavage reaction is around 7.4. With a common used peptide substrate, its optimal pH is artificially increased to higher than 9.
pH Profiles of Adam17 to its native protein substrate and to a peptide substrate
Substrate-bound Protease Inhibitors
Our assay provides an essential tool for discovery of inhibitors as well as monoclonal antibodies bind to the cleavage site of substrate protein. Historically, protease drug development has been focused on the active site inhibitors that bind to the enzyme. This strategy is facing increasing challenge due to selectivity issues. First, proteases of the same family share the similar mechanisms and structures at their active sites. As a result, an active site inhibitor often shows a broad inhibition profile to the proteases family. This non-specific effect is proven to be difficult to overcome because it originates from the structure and mechanism of the target. Second, a protease often has multiple substrates in cells, each links to different downstream physiological effect. Inhibition of such a protease inevitably blocks all functions regulated by the enzyme. Apparently, these two selectivity problems do not exist for a substrate-bound inhibitor. Since a substrate inhibitor does not interact with the enzyme, it would neither affect the cleavage to other substrates of the target protease, nor would the activity of the similar proteases. Therefore, substrate inhibitor is an attractive approach for development of highly selective protease drugs that have been difficult for the traditional active site inhibitors. This is particular promising for monoclonal antibody drugs.
Other Application
  •    Leads optimization and SAR based on physiological relevant reaction
  •    Development of antibody drug targeting substrate proteins
  •    Kinetic and mechanism studies of protease reaction to its natural substrate
  •    Determination of pH, salt concentration and other factors affect protease reaction
  •    Fluorescent image of substrate protein without interference of bulky fluorescent protein
Reference
Publication is available online
Analytical Biochemistry. To request a reprint, please email us service@ezbiolab.com.