“Cloning and sequencing of four cysteine porteases from RNA extracted from latex of plants grown in Argentina”

TREJO, S.A.; SEQUEIROS, C., TORRES M.J., MARTÍN M.I., LÓPEZ, L.M.I, AVILÉS F.X. AND NATALUCCI C.L.

Portoroz, Slovenia

Conferencia; V International Conference on Cysteine Proteases and their Inhibitors: from structure to regulation and biology.; 2006

Latex is a good source of proteases, specially cysteinendopeptidases. We have previously purified and characterized several cysteine proteases from latex of Asclepias fruticosa, Philibertia gilliesii (Apocynaceae), and Carica quercifolia (Caricaceae), which differ in pI values as well as molecular weight and biochemical characteristics. The aim of this work was to clone, sequence, and compare the cDNA of cysteine proteases expressed in latices of the mentioned species. Total RNA was extracted from latex and used for retro-transcription. Degenerate oligonucleotides for using in amplification reactions were designed according to N-terminals previously sequenced. The cDNAs were submitted to PCR, and the amplification products corresponding to the cDNA full length (approximately 0.6-0.8 kb) were cloned using pGEM-T Easy (Promega) vector and XL1 Blue E. coli strain. DNAs extracted from plasmids were sequenced and translated, and consensus sequences were obtained for four cysteine proteases. The four sequences were homologous each other and also with other different plant cysteine proteases belonging to the papain family. In addition, highly conserved domains characteristic of this group of endopeptidases can be identified in these sequences. However, some differences in the primary sequence indicate that these proteases could have distinctive characteristics either in specificity as in other biochemical aspects. It should be noted that no previous references exist about protease sequences obtained from RNA of latex.