Proteases have proven to be excellent drug targets. Based on the experiences from angiotensin converting enzyme to HIV protease inhibitors, many therapeutics will be targeted to proteases found from analysis of microbial and human genomes. Although BLAST analyses will often place a candidate gene within certain families (e.g., aspartyl proteases), it is still sometimes difficult to find satisfactory surrogate substrates a priori. A powerful approach to rapidly discover substrates is described here (7). It is based on the principle of Fluorescent Resonance Energy Transfer, FRET (Fig. 1). In this case, Lucifer yellow is paired with Dabsyl in a FRET quenching strategy to discover peptide sequences that can be cleaved to disrupt the FRET quenching phenomenon, and the beads with such substrates become fluorescent. We expect that the scope of this approach should include other hydrolases and substrate classes amenable to combinatorial methods and structural determination. The method described here uses a purified protease for screening.
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