Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae)

• Published in: Phytochemistry (Oxford) Vol. 81; pp. 7 - 18
• Main Authors: Lufrano, Daniela, Faro, Rosário, Castanheira, Pedro, Parisi, Gustavo, Veríssimo, Paula, Vairo-Cavalli, Sandra, Simões, Isaura, Faro, Carlos
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.09.2012
• Subjects: amino acids; Aspartic Acid Endopeptidases - chemistry; Aspartic Acid Endopeptidases - genetics; Aspartic proteinase; aspartic proteinases; Asteraceae; Bull thistle; Caseins - chemistry; cheesemaking; Cirsin; Cirsium - chemistry; Cirsium - enzymology; Cirsium - genetics; Cirsium vulgare; clones; Cloning, Molecular; complementary DNA; DNA, Complementary - chemistry; DNA, Complementary - genetics; Enzyme Activation; Enzyme Assays; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; flowers; Flowers - chemistry; Flowers - enzymology; Flowers - genetics; Hydrogen-Ion Concentration; milk clotting; Milk-clotting activity; molecular cloning; Pepsin-like protease; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Procirsin; Proteolysis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; rennet; Sequence Homology, Amino Acid; Substrate Specificity; taxonomy; vegetables; Pepsin-like protease; E-64; PSI; MCA; EDANS; EDTA; DABCYL; EPNP; Cirsium vulgare; AP; Procirsin; Aspartic proteinase; Asteraceae; SAPLIPs; Bull thistle; Milk-clotting activity; Cirsin
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2012.05.028

Recombinant procirsin displayed a high specificity towards κ-casein and milk-clotting activity suggesting its potential use as a new milk coagulant. [Display omitted] ► cDNA encoding an aspartic protease precursor was isolated from Cirsium vulgare flowers. ► The encoded protease (cirsin) shares with cyprosin a striking sequence similarity. ► Recombinant procirsin was active without undergoing any activation process. ► This precursor form displayed the typical proteolytic properties of aspartic proteases. ► Recombinant procirsin also cleaved κ-casein and displayed milk-clotting activity. Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.