Production and characterization of a recombinant alpha-N-acetyl glucosaminidase enzyme in the Pichia pastoris yeast

N-acetyl-alpha-glucosaminidase (NAGLU) is an homodimeric lysosomal enzyme encoded by the NAGLU gene. Mutations in this gene cause the loss of enzyme activity and the accumulation of heparan sulfate (HS) in lysosomes. This deficiency is the cause of mucopolysaccharidosis type IIIB (MPSIIIB) or Sanfilippo syndrome type B, for which the clinical symptoms are mainly associated with the central nervous system resulting in mental retardation and hyperactivity. Currently, there is no approved therapy for this disorder. Enzyme replacement therapy (ERT) has been proposed as a potential treatment alternative, for which recombinant enzymes have been produced in mammalian cells. However, other expression systems such as yeasts may be used as host due to their ability to produce N-glycosylations like those observed in human proteins. In this study we evaluated the production of recombinant NAGLU in the yeast Pichia pastoris GS115 as an expression system. The strain was transformed with the NAGLU gene inserted into the plasmid pPICZ-α, which expressed NAGLU under the control of AOX1 promoter. Ten clones were selected and the insertion of the gene was confirmed by PCR. Positive clones were scaled up to 100 and 400 mL. Two clones showed the highest enzyme activities values between 0.074 and 0.121 U/mL at 100 ml after 72 h of induction. The best clone was produced at 400 mL showing an enzyme activity of 0.187 U/mL at 72 h. The recombinant enzyme was purified using affinity chromatography column and verified by SDS-PAGE and Western blot. The purified enzyme was characterized in terms of temperature and pH stability, as well as by its ability to be taken up by MPS IIIB patient fibroblasts. These results show the potential of P. pastoris as an expression system for the production of recombinant human NAGLU, which can allow the development of an ERT for MPS IIIB.