Cloning and Sequencing of Cystein Proteases from Latex of Philibertia gilliesii

SEQUEIROS, CYNTHIA; TREJO, SEBASTIÁN A.; DIONISI, HEBE M.; AVILÉS, XAVIER F.; NATALUCCI, CLAUDIA L

Pinamar, Buenos Aires, Argentina

Congreso; Xth Congress PABMB and 41st Annual Meeting SAIB; 2005

Panamerican Association for Biochemistry and Molecular Biology - Sociedad Argentina de Investigación Bioquímica y Biología Molecular

We have previously determined the presence of several cysteine proteases in the latex of P. gilliesii by biochemical studies. They differ in their pI as well as their molecular weight. The most alkaline of these endopeptidases (philibertaine g I) was purified, and its Nterminal sequence was obtained. The aim of this work was to clone and sequence the cDNA of cysteine proteases expressed in the latex of P. gilliesii. We designed degenerate primers based on the Nterminal sequence of philibertaine g I, extracted total RNA from latex, obtained cDNA (about 0.8 kb) by RT-PCR, and cloned these products into pGEM-T Easy Vector (Promega). At least two different cysteine proteases were identified within the sequenced clones. Deduced amino acid sequences from both clones showed similarities of 64 and 56% with papaya proteinase omega (CAA49504). In addition, highly conserved domains characteristic of these endopeptidases can be identified in both groups of sequences. However, none of the obtained sequences matched the N-terminal sequence of philibertaine g I. Therefore, latex of P. gilliesii can be a promising source of novel proteases with potential technological applications.