Abstract

Xyloglucan endotransglycosidase/hydrolase (XTH) enzymes participate in hemicellulose dissembling required for cell wall remodeling, by means of its endotransglycosidase (XET; EC 2.4.1.207) and/or hydrolase (XEH; EC 3.2.1.151) activities. Previous studies have described the participation of XTHs during softening of Fragaria chiloensis fruit. XET and XEH activities, and the presence of XTH protein, have been detected in F. chiloensis fruit at the turning stage. Furthermore, two full-length cDNA sequences, FcXTH1 and FcXTH2, have been isolated, showing the two isoforms a differential tissue specific expression profile. Interestingly, FcXTH1 is highly expressed in fruit, mainly at the large green and turning stages, when major significant fruit firmness changes are taking place. In this study, we present the molecular cloning of FcXTH1 and the heterologous expression of FcXTH1 protein. The gene was cloned in Escherichia coli and then subcloned and expressed in Pichia pastoris. The recombinant protein was secreted to the yeast culture and then purified through a His-tag affinity chromatography. The recombinant protein was detected by immuno-blotting, evidencing a size of ~44 kDa. In silico analysis predicted a lower molecular weight protein (~35 kDa). Additionally, the FcXTH1 amino acid sequence presents putative N-glycosylation sites, which suggests that these post-translational modifications might explain the differences in molecular weight between prediction and experimental results. The activity of the recombinant protein and the effect of N-glycosylation for activity are under study.