Production and characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli BL21

Morquio A disease (MPS IVA) is an autosomal recessive disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) that leads to the accumulation of keratan sulfate and chondroitin-6-sulfate mainly in bone and cornea. Previously, we showed the feasibility for producing an active recombinant GALNS (rGALNS) enzyme in E. coli BL21 at shacked and bench scales. In this work, we report the production of a rGALNS at bench scale by using a fed-batch strategy, and the purification and characterization of the recombinant enzyme. This strategy allowed a 5-fold increment in enzyme production in the crude protein extract from cell lysate compare to those levels observed with a batch strategy (0.05 U/mg vs 0.01 U/mg). Noteworthy, recombinant enzyme was detected in culture media from fed-batch cultures; with enzyme activities of up to 2.9 U/mg. rGALNS was purified by anion exchange and gel filtration chromatography. Enzyme stability of the purified enzyme both from cell lysate and culture medium was characterized at pH from 3.0 to 8.0 at 4, 37 and 45 °C. Finally enzyme uptake was evaluated in HEK293 cells and human MPS IVA fibroblasts. In summary, these results constitute valuable evidence towards the development of an enzyme replacement therapy for MPS IVA using a prokaryotic recombinant enzyme.