Production of an active recombinant human N-acetylgalactosamine-6-sulfate sulfatase enzyme in Pichia pastoris

Currently, Morquio syndrome type A patients lack of a specific treatment, and enzyme replacement therapy (ERT), using an enzyme produced in CHO cells that is currently in clinical trials, represents the closest treatment option for these patients. As an alternative to this enzyme, we report the production and characterization of a recombinant GALNS using the methylotrophic yeast P. pastoris. Human GALNS and SUMF1 cDNA were subcloned separately into the pPIC9 plasmid and used to co-transformed P. pastoris GS115. Production was carried out 1.65 L scale, and recombinant enzyme was purified from the conditioned medium. Purified enzyme was used for temperature and pH stability analysis, and cell uptake assays. GALNS activity at the crude extract was 0.2 U/mg, which was increased up to 7.5-fold with SUMF1 co-expression. Western-blot analysis showed the presence of a 120 kDa protein, which was processed to 50 and 30 kDa peptides. Recombinant enzyme was purified 979-fold with 33% yield and an activity of 25.3 U/mg, which is higher that the activity reported for recombinant GALNS produced in CHO cells. Recombinant GALNS showed a maximum activity at pH 5.0 and was stable for at least 48 h at 4 °C, although rapidly lost activity at 37 °C. Noteworthy, recombinant GALNS was taken up by HEK293 cells and human MPS IVA fibroblasts, suggesting that P. pastoris glycosylations might be recognized by human receptors. In conclusion, these results show the potential of the recombinant GALNS produced in P. pastoris for the development of an ERT for Morquio syndrome type A.