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

Morquio disease type A is produced by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Recently we reported the production and characterization of a recombinant GALNS (rGALNS) in E. coli. Although we obtained a catalytically active enzyme, the lack of N-glycosylation prevented cellular uptake of the recombinant protein. In this study, we evaluated the production of rGALNS in the methylotrophic yeast Pichia pastoris as an alternative to produce a recombinant glycosylated protein in a microorganism. Human GALNS cDNA, using the native or a heterologous signal peptide (SP), was subcloned into pPIC9 plasmid and transformed in P. pastoris GS115. In addition, P. pastoris was co-transformed with GALNS cDNA and human SUMF1 cDNA. Production was carried out at 0.01, 0.1, and 1.5 L scales. As expected, GALNS activity was detected extracellulary, while intracellular enzyme activity was not observed. Use of heterologous SP (− Factor), allowed higher enzyme activity levels than when the native SP was used. The rGALNS activities were up to 0.02 and 0.09 U/mg for the strains with native or -Factor SP, respectively, which were higher than those levels obtained extracellulary for batch cultures of E. coli. Co-expression with SUMF1 allowed a 2-log-fold increment in enzyme activity, showing the advantage of sulfatase-SUMF1 co-expression within a yeast system. ELISA quantitation showed a yield of production of up to 130 μg/L. In summary, these results show for the feasibility for the production of rGALNS enzyme in P. pastoris. Current studies are focus in purification, characterization, and cell uptake of this recombinant GALNS enzyme.