Human lysosomal β-N-acetylglucosaminidases produced in Pichia pastoris GS115: a feasible source for enzyme replacement therapy

Tay-Sachs and Sandhoff diseases are two lysosomal disorders caused by the deficiency of the lysosomal hexosaminidases Hexo-A and Hexo-B, which are dimeric enzymes expressed by different genes. These diseases lack a specific therapy; enzyme replacement therapy (ERT) is a feasible option for these patients. Proteins used for the ERT of lysosomal disorders are currently obtained from recombinant mammal cells. Nevertheless, other expression systems, such as microorganisms and plants, have been evaluated to produce recombinant lysosomal proteins for therapeutic purposes. In this study we produced recombinant Hexo-A, Hexo-B and Hexo-S in the methylotrophic yeast Pichia pastoris. The genes encoding these proteins were optimized for P. pastoris and subcloned independently into pPIC9K plasmid. Production of recombinant enzymes was evaluated extracellularly. At 1.65 L the highest specific enzyme activities obtained were 12,624 U/mg, 10,343 U/mg, and 14,606 U/mg for rhHexo-A, rhHexo-B and rhHexo-S, respectively. These results are about 25- to 30 fold higher that those obtained in normal human leucocytes. Recombinant enzymes were purified by ion exchange and affinity chromatography. These are being used in cellular uptake and in vitro interactions evaluation assays. These results show the potential of P. pastoris GS115 for the production of recombinant hexosaminidases to be used in ERT.