Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates

Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor-processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage.

Automated summary

Proteases play a crucial role in controlling plant growth and development by limited proteolysis, including the processing of peptide hormone precursors. Ascertainment of candidate protease as a peptide precursor-processing enzyme requires in vitro demonstration of protease-mediated precursor cleavage. In this study, we offer a protocol for the expression, purification, and characterization of tomato phytaspases as candidate proteases for phytosulfokine precursor processing, and we also demonstrate the use of synthetic oligopeptide substrates for site-specific precursor cleavage.