Characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli for enzyme replacement therapy of Morquio A disease

• Published in: Process biochemistry (1991) Vol. 47; no. 12; pp. 2097 - 2102
• Main Authors: Mosquera, Angela, Rodríguez, Alexander, Soto, Carlos, Leonardi, Felice, Espejo, Angela, Sánchez, Oscar F., Alméciga-Díaz, Carlos J., Barrera, Luis A.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01-12-2012
• Subjects: arylsulfatase; blood serum; chromatography; cultured cells; enzyme stability; Escherichia coli; fibroblasts; GALNS; glycosylation; humans; Morquio A; mucopolysaccharidosis; N-linked oligosaccharides; oligosaccharides; patients; temperature; therapeutics; GALNS; Morquio A; N-linked oligosaccharides; Escherichia coli
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2012.07.028

► rGALNS was extracellularly produced in Escherichia coli BL21. ► Purified enzyme showed a 0.21Umg−1 activity and a production yield of 0.78mgL−1. ► rGALNS showed similar temperature and pH stability profiles to mammalian GALNS proteins. ► rGALNS was not taken up by culture cells. ► Glycosylations seem to be necessary for protein uptake but not for stability.
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Currently, specific therapies are not available for MPS IVA patients. In this study, a biologically active recombinant GALNS enzyme (rGALNS) produced in Escherichia coli was purified through a two-step chromatography process. The effect of temperature and pH on purified rGALNS stability was evaluated, as well as the stability in human serum. Finally, the uptake of rGALNS by HEK 293 cells and MPS IVA fibroblasts was evaluated. The use of a semi-continuous process allowed the production of an active extracellular rGALNS, which was used for protein purification. The purified rGALNS showed a specific activity of 0.29Umg−1 and a production yield of 0.78mgL−1. The rGALNS presented an optimal pH of 5.5 and was stable for 8 days at 4°C. In human serum it was stable for up to 6h. rGALNS was not taken up by the cultured cells, suggesting that N-linked oligosaccharides are not necessary for the production of an active enzyme or enzyme stability but for the cell uptake of protein. This study shows the first characterization of rGALNS produced by E. coli, and provides important information about purification, stability, and glycosylations effect for this type of enzymes.