Production of Functional Plant Legumain Proteases Using the Leishmania tarentolae Expression System.

Plant proteases of the legumain-type are key players in many processes along the plant life cycle. In particular, legumains are especially important in plant programmed cell death and the processing and maturation of seed storage proteins within the vacuole. Plant legumains are therefore synonymously called vacuolar processing enzymes (VPEs). Because of their dual protease and cyclase activities, plant legumains are of great interest to biotechnological applications, e.g., for the development of cyclic peptides for drug design. Despite this high interest by the scientific community, the recombinant expression of plant legumains proved challenging due to several posttranslational modifications, including (1) the formation of structurally critical disulfide bonds, (2) activation via pH-dependent proteolytic processing, and (3) stabilization by varying degrees of glycosylation. Recently we could show that LEXSY is a robust expression system for the production of plant legumains. Here we provide a general protocol for the recombinant expression of plant legumains in Leishmania cells. We further included detailed procedures for legumain purification, activation and subsequent activity assays and additionally note specific considerations with regard to isoform specific activation intermediates. This protocol serves as a universal strategy for different legumain isoforms from different source organisms.