Identification of artichoke aspartic proteases using proteomics tools

LUFRANO, D.; OBREGÓN, W.D.; LIGGIERI, C.; TREJO, S.; VAIRO CAVALLI, S.

Rosario, Santa Fe, Argentina

Congreso; VII Simposio Nacional de Biotecnología y II Congreso Internacional-REDBIO-Argentina; 2009

REDBIO-Argentina

Cyprosin, cardosin A and cardosin B, aspartic proteases extensively studied, are mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe´s cheese. Milk clotting activity is due to the presence of these peptidases. Cardosins and cyprosins are model plant aspartic proteases, a group of proteases that are involved in cell death events associated with plant senescence and stress responses. They are synthesized as single-chain zymogens, and subsequent conversion into two-chain mature enzymes is a crucial step in the regulation of their activity. Cardosin and cyprosin genes coexist in the genome of the same plant. Extract of Cynara scolymus, L. (artichoke) flowers have been investigated as a source of enzymes to be used in cheese making as an alternative or in addition to calf rennet. In this work, we report the proteomic analysis of the two major proteases present in artichoke’s flowers. Proteases were purified by anion-exchange chromatography on DEAE-Sepharose. The main fractions (4 and 5) were separated by SDS-PAGE. Each enzyme presented two subunits of approximately 30 and 15 kDa. Bands were cut and then subjected to tryptic digestion. The resulting peptides were analyzed by MALDI-TOF spectrometry. PMFs spectra were interpreted with the MASCOT software. The major subunit (30 kDa) of fractions 4 and 5 match to cardosin H, a minority of C. cardunculus proteases .