Process Biochemistry 47 (2012) 2097–2102 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u rn al hom epage: www.elsevier.com/locate/procbio Characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli for enzyme replacement therapy of Morquio A disease Angela Mosquera a,1 , Alexander Rodríguez a,1 , Carlos Soto a , Felice Leonardi b , Angela Espejo a , Oscar F. Sánchez b,∗,2 , Carlos J. Alméciga-Díaz a,∗∗ , Luis A. Barrera a a Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia b Chemical Engineering Department, Universidad de Los Andes, Bogotá, Colombia a r t i c l e i n f o Article history: Received 11 May 2012 Received in revised form 25 June 2012 Accepted 23 July 2012 Available online 31 July 2012 Keywords: Morquio A GALNS Escherichia coli N-linked oligosaccharides a b s t r a c t 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 0.29 U mg -1 and a production yield of 0.78 mg L -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 6 h. 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. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease, OMIM #253000) is an autosomal recessive disease caused by the deficiency of the lysosomal enzyme N-acetyl-galactosamine- 6-sulfate sulfatase (GALNS, EC 3.1.6.4) [1]. Deficiency of GALNS prevents the normal degradation of keratan- and chondroitin-6- sulfate causing their lysosomal accumulation [1,2]. Clinical features of MPS IV A patients include marked short stature, hypoplasia of the odontoid process, pectus carinatum, kyphoscoliosis, genu val- gum, laxity of joints, and corneal clouding, without central nervous ∗ Corresponding author at: Department of Chemical Engineering, Universidad de Los Andes, Kra 1 E No. 19 A-40, Bogotá, Colombia. Tel.: +57 1 339 4949x2799; fax: +57 1 332 4334. ∗∗ Corresponding author at: Laboratory for Proteins Expression and Purification, Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Kra 7 No. 43-82, Building 53, Room 303A. Bogotá, Colombia. Tel.: +57 1 320 8320x4140; fax: +57 1 320 8320x4099. E-mail addresses: ofsanchezm@unal.edu.co (O.F. Sánchez), cjalmeciga@javeriana.edu.co (C.J. Alméciga-Díaz). 1 These authors contributed equally to this work. 2 Current address: Pharmacy Department, Universidad Nacional de Colombia. Bogotá, Colombia. system impairment [1,3]. Life span is variable and depends on the spectrum of disease severity, but most patients die between the second and third decade of life [1,3,4]. At present, there is not specific treatment for this disease, and it is limited to supportive measures and surgical interventions to alleviate some manifestations of the disease [1,3]. Although bone marrow transplantation is the best treatment option for some mucopolysaccharidosis, it has a limited benefit in MPS IV A patients besides the risk of fatal complications [5]. Gene therapy, widely explored in animal models for almost all mucopolysaccharidosis types [6], has shown to be a feasible alternative for the treatment of Morquio A disease [7–9], albeit it is still in early stages. Currently, the main treatment option for lysosomal storage dis- orders is the enzyme replacement therapy (ERT). Gaucher, Fabry, Hurler (MPS I), Hunter (MPS II), Maroteaux–Lamy (MPS VI), and Pompe diseases have approved ERT, and it has shown an improve- ment of some systemic manifestations of diseases [10]. In the case of ERT for MPS IVA, preclinical trials of ERT using recombinant enzymes have shown significant decrease of KS in blood and tissues [11,12] or in cultured cells [13]. Human GALNS is a homodimeric protein of 522 amino acids, composed of 60 kDa monomers processed to peptides with of 40 and 15 kDa [14,15]. Native GALNS has been purified from liver [16] and placenta [17], and produced in recombinant Chinese hamster 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.028