Profiling of Cancer Related Protease

Proteases are enzymes that break apart proteins. They cleave peptide bonds between specific amino acid, depending on the specialized proteases. Some proteases degrade collagen, which is part of extracellular matrix, and this degradation allows cell growth. We are interested in studying proteases that have been implicated in cancer, and utilize a new developing method for protease characterization, called cellular libraries of peptide substrates (CLiPS), to investigate the specificity of cancer-related proteases.

Peptides produced by bacteria are displayed on the bacteria cell surface. The peptide includes a ligand region, which will bind to the fluorescent probe and a substrate region, which will be cleaved if treated with proteases. When the substrate is cleaved, the peptide ligand region would also be removed from the cell; therefore, the cell would be non-fluorescent. We utilize fluorescent-activated cell sorter (FACS) to detect the presence of intact substrates.

We found out that removing all the growth media before protease incubation is a very important step. This step reduces cell growth during the reaction step, and leads to more repeatable results. High cleavage conversion rate samples provide us the target peptide sequences and this is how we find the optimum peptide sequences for the specific proteases. Both reaction time and protease concentration are contributing factors to getting high cleavage conversions. Shortening reaction time gives us various and lower conversion rates. The higher the conversion rates for a given reaction time, the more optimal the peptide sequences.