Characterizing Protease Activity and Selectivity
Proteases are responsible for cutting peptide bonds in proteins and regulate many biological processes including cell growth and migration, blood coagulation, and programmed cell death. Protease misregulation is common to many diseases such as cancer, therefore it is important to understand which proteins in the body can be easily cut by a specific protease. The goal of this project is to determine what substrates are commonly cut by a given protease and to design inhibitors that can block protease activity and reduce disease symptoms. During the summer, we have characterized the activity of the protease human kallikrein 7, by first producing reporter substrates capable of Förster resonance energy transfer (FRET) using bacteria. We set up an enzymatic assay in a 96 well plate by mixing a premade solution containing the protease human kallikrein 7 with six FRET reporter proteins that each contained a different substrate. By measuring the change in fluorescence over time, we were able to determine how fast our protease cut the different substrates and hypothesize why certain substrates are cut faster than others.