Purification of Phytaspases Using a Biotinylated Peptide Inhibitor.

Abstract

Phytaspases are plant subtilisin-like proteases (subtilases) that possess a rarely occurring substrate cleavage specificity. These proteolytic enzymes hydrolyze their substrates strictly after an aspartate residue preceded by a characteristic, though degenerate, tripeptide amino acid motif. Having been initially discovered as proteases involved in the accomplishment of programmed cell death in plants, phytaspases were also demonstrated to be instrumental in specific processing of precursor proteins of several plant peptide hormones, thus generating biologically active peptides. Here we provide a protocol for isolation of active phytaspases from leaves, which was shown to be efficient for a wide range of plant species. The key element of the proposed scheme is the use of a specific and reversible biotinylated peptide aldehyde inhibitor of phytaspases for purification of the enzymes by means of affinity chromatography. We also discuss nuances, pitfalls, and possible alternatives for successful isolation of proteolytically active phytaspases.