Production of human recombinant alpha-N-acetylglucosaminidase enzymes in two Pichia pastoris strains

Lysosomal human hexosaminidases (alpha-N-acetylglucosaminidase) are a group of three dimeric isozymes expressed by different genes. HexoA is a heterodimeric protein (αβ) while HexoB (ββ) and HexoS (αα) are homodimeric. Mutations in either of the subunits cause Tay–Sachs or Sandhoff diseases. In this study we produced recombinant HexoA and HexoB isozymes in two strains of the methylotrophic yeast Pichia pastoris. The subunits genes were optimized for P. pastoris expression and native signal peptide were removed and replaced with the signal secretion from Saccharomyces cerevisiae. The genes were subcloned into pPIC9K plasmid for the P. pastoris GS115 strain and into pPink-HC plasmid for the PichiaPinkTM strain. Production of recombinant enzymes was first carried out at 10 mL lot and the best clones were then produced in scaled-up lots of 100 mL. Total protein concentration, biological activity, SDS-PAGE, and western blot in extracellular samples were evaluated. The highest specific enzyme activities obtained were 1205 and 276,031 U/mg in PichiaPinkTM, as well as, 1837 and 137,911 U/mg in P. pastoris GS115 for rhHexoA and rhHexoB, respectively, obtaining more than 3-fold and almost 400-fold in comparison to HexA and HexB leucocytes activity. Crude extracts were concentrated and used to evaluate cell uptake of the recombinant enzymes by using HEK293 cells. These results show the potential of P. pastoris as a platform for the production of active recombinant hexosaminidases as an option for enzyme replacement therapy. Further studies should consider the scaling up of the process, purification, glycosylation studies and the in vivo evaluation of the enzymes.