Expression and characterization of glucocerebrosidase in lineages of p. pastoris with different patterns of glicosilation

Abstract

The production of recombinant proteins is a recorrent approach for comercials and farmaceutic purposes. Among the heterologous systems, the yeast Pichia pastoris is one of the most explored systems because of its high production, beeing a advantageous choice for the proejct. The deposition disease studied in the research, Gauchers disease, occurs because of the absense or deficiency of the glucocerebrosidase enzyme (GCase), causing accumulation of glucocerebroside and formation of Gauchers cells, wich cause the disease symptons. The treatment is the enzymatic repositioning of this enzyme, wich is produced in genetically enginereed Chinese hamster ovary (CHO) cells and distributed by SUS. The import of this medicine cause a expensive cost for public health. The objective of this project is the production of the GCase in P. pastoris, with expression vectors for transformation of comercial wild yeast and the P. pastoris GlycoSwitch lineage for expression, purification and comparation of the produced protein in both systems. The comercial wild P. pastoris should result in a hypermanosylated GCase, while the GlycoSwitch lineage should result in a GCase with similar glycosylation pattern to the comercial product Cerezyme (Genzyme,from the company Sanofi-Pasteur).