Cloning and sequencing of a papain-like proteinase obtained from a South American native plant

OBREGÓN, D.; TREJO, S.; MORCELLE, S.; BRONSOMS, S.; LLERENA, C.; PRIOLO, N.; AVILÉS, F.X.

Bilbao, España

Congreso; XXXI Congreso de la Sociedad Española de Bioquímica y Biología Molecular; 2008

Sociedad Española de Bioquímica y Biología Molecular

The cDNA belonging to a protease from fruits of Araujia angustifolia was obtained by RT and 3´ RACE-PCR methods after total RNA extraction. The cDNA fragments obtained by PCR (of approximately 760 bp) were cloned into E. coli XL1-Blue strain using the pGEM-T Easy vector (Promega) and the clones were sequenced. A consensus sequence of 760 bp was derived from the ClustalX analysis of the sequenced clones. The consensus sequence codifies for a 214-residue mature peptidase named araujiain aII containing seven cysteine residues that are highly conserved in papain-like peptidases: Cys25 belongs to the peptidase active site, whereas the other six cysteines are involved in three disulfide-bonds formation that are characteristic of this class of enzymes (Cys22-Cys56, Cys63-Cys95, Cys150-Cys201). Blast analysis of the predicted protein sequence of araujiain aII showed significant homology with other cysteine plant proteases as caricain, actinidain, chymopapain and papain. They share approximately 50% of sequence identity and display highly preserved regions, as those belonging to the catalytic site (Gln19, Cys25, His159 and Asn175).