Cloning and expression of N-Ac Glucosaminidase of Xenopus laevis

MORALES, E.; ARRANZ, SILVIA EDA; CABADA, M.

Rosario

Congreso; XLII Reunion Anual 42TH Annual Meeting; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Sperm-egg recognition events involve carbohydrate-protein interactions in mammals. Among amphibians, sperm binding in Xenopus laevis is mediated at least by N-Ac Glucosamine residues of the vitelline envelope (VE). We have shown that N-Ac glucosaminidase is the most important glycosidase activity in Bufo arenarum sperm and that binds to components of the VE. The aim of the present work was to produce a specific N-Ac Glucosaminidase antiserum as a prerequisite for its biochemical and functional characterization, since antibodies of other animals are not available. By in-silico searching on EST?Ls from Xenopus laevis embryos, we identified clones codifying for the cDNA of the enzime that were gently provided by the National Institute of Basic Biology of Japan. A sequence with the complete ORF was subcloned in four different bacterial expression vectors and the resulting product shown to be toxic for E. coli in all the cases. To overcome this problem, a sequence coding for an amino terminal fragment (18.6 kDa) of the enzime was cloned and the expression conditions optimized. The recombinant protein, GST fused, was purified with Glutathione-Sepharose followed by SDS-PAGE and electroelution, and used to produce antibodies in rabbits. The antiserum (titer 1/750 for 0.15 ƒÊg of the fusion protein) specifically recognized the recombinant protein but not GST.