Cloning and sequencing of a cysteine protease from latex of Vasconcellea quercifolia (Caricaceae)

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

Patras, Grecia

Congreso; 5th General Meeting of the International Proteolysis Society; 2007

Sociedad Internacional de Proteolisis

The latex of Vasconcelleae quercifolia A. St.-Hil. contain several cysteine endopeptidases with very high proteolytic activity, which are the active compounds used by the plant as a defence mechanism against herbivorous insects. In contrast to the cysteine proteinases present in the latex of Carica papaya (Caricaceae), which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. Our group has previously isolated and characterized two basic cysteine proteases from the latex of V. quercifolia. The aim of this work was to clone and sequence the cDNA of cysteine proteases expressed in V. quercifolia latex. Total RNA was extracted from latex with the NucleoSpin RNA Plant Kit (BD Biosciences) and used for retro-transcription. Degenerated oligonucleotides for using in amplification reactions were designed according to N-terminal peptides of another cysteine plant protease previously sequenced from Asclepias fruticosa latex. The cDNA was submitted to PCR and the amplification products corresponding to the cDNA full length (approximately 0.6-0.8 kb) were detected in 1% agarose gels with ethidium bromide. Bands were extracted with QIAEX II Gel Extraction kit (Qiagen), PCR amplified and cloned using pGEM-T Easy vector (Promega) and XL-1 Blue E. coli strain. The plasmidic DNA was translated using the Translate Tool (ExPASy) software. The consensus sequence (VqCP-A) was aligned using the Blast network service showing a high similitude degree with different plant cysteine proteases belonging to the papain family. The full amino acid sequence contains 211 aminoacidic residues, the conserved active site amino acid residues Gln19, Cys25, His156 and Asn176 (papain numering) and highly conserved domains characteristic of these group of endopeptidases. Some physicochemical properties of VqCP-A were obtained using the GPMAW software (MW = 23449.3 Da, pI = 9.43, extinction coefficient = 49765 M-1 cm-1).