A Novel Genetically Engineered Protein Module for a Qd-Based FRET Biosensor

Proteases are involved in many essential cellular processes such as blood coagulation, fibrinolysis, hormone maturation, and apoptosis. They are also used as the key virulence factors for pathogenic infection. These properties make proteases a prime target for detailed investigation to better understand the disease development process and to identify targets for drug treatment.

One of the most promising methods for probing protease activity is based on the principle called fluorescence resonance energy (FRET) containing two fluorophores located less than 100 Ǻ apart. In this study, we develop a genetically engineered protein module that is designed with 1) a quantum dot (QD)-binding moiety containing poly-histidine, 2) a specific site for fluorescent dye incorporation based on unnatural amino acid, 3) a protease cleavage site, 4) a flanking TAT peptide sequence for cell penetration and 5) an elastin-like protein (ELP) domain for thermal purification. We will investigate the use of this new genetically programmable protein module as QD-based FRET substrates for protease inhibitor discovery. The advantages of this approach include the significant reduction of cost on peptide synthesis, easy modulation of the placement of unnatural amino acid and intracellular monitoring of protease activity.