Cloning and sequencing of four cysteine proteases 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; International Conference on Cysteine Proteinases and Their Inhibitors: From structure to regulation and biology; 2006

The latex is a good source of proteases, specially cysteinendopeptidases. We have previously isolated and characterized several cysteine proteases from latex of Asclepias fruticosa, Philibertia gilliesii (Apocynaceae), and Carica quercifolia (Caricaceae). They differ in their pI as well as their molecular weight and biochemical characteristics. The aim of this work was to clone, sequence, and compare the cDNA of cysteine proteases expressed in those latex. Total RNA was extracted from latex and used for retro-transcription. Degenerated 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 detected in agarose gels with ethidium bromide. Bands were extracted with the QIAEX II kit (Qiagen), PCR amplified and cloned using pGEM-T (Invitrogen) vector and XL1 Blue E. coli strain. From plasmidics DNA were translated and consensus sequences were obtained for four cysteine proteases. The four sequences were homologous and were homologous too with other different plant cysteine proteases belonging to the papain family. In addition, highly conserved domains characteristic of these group of endopeptidases can be identified in these sequences. However, some differences in the primary sequence indicate that these proteases have distinctive characteristics either in specificity as in other biochemical aspects. Our works have been first in which it has been possible to obtain the proteases sequence from RNA of latex.