Creation of a recombinant peptide substrate for fluorescence resonance energy transfer-based protease assays.

The performance of protease assays has been revolutionized by the advent of Xuorescence resonance energy transfer (FRET)-based assays. Generation of pure, reliable, high-quality substrate is essential to such assays. Small FRET substrates can be easily prepared by chemical production. The preparation of Xuorogenic substrate becomes cumbersome when a larger peptide substrate is required or when the amino acid residues that Xank the protease cleavage site are critical to enzyme activity. We sought a novel method to generate a large FRET peptide substrate using facile DNA recombinant technology. FRET is a phenomenon that describes the alteration in Xuorescence emission that occurs with the interaction of two molecules in close proximity. Energy transferred from an excited donor molecule (Xuor) to an acceptor molecule (quencher) is dependent on the Förster radius (R0). This is the distance at which 50% eYcient energy transfer occurs between these two molecules. Typical Förster radii are in the range of 30–50 Å [1]. The FRET rate is inversely proportional to the sixth power of the distance between the donor and the acceptor [2]. Two diVerent approaches have been used to develop FRET protease assays based on minimal eVective recognition sequence. Enzymes that have speciWcity deWned by the residues amino to the cleavage site of the substrate (P residues) can be assayed using substrates containing a Xuorescent leaving group at the carboxy terminus and a quenching group on the short peptide. Enzymes with critical residues carboxy to the cleavage site (P residues) require a second approach. Substrates