Human recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in a glycoengineered Escherichia coli strain

In this project, recombinant GALNS (rGALNS) was produced in E.colieither with native or codon-optimized gene. Codon-optimized gene allowed higher production of rGALNS. In addition, rGALNS wasproduced in E. coli BL21(DE3), which has no N-glycosylation machinery, and in E. coli MC4100, which was transformed with aplasmid expressing the genes agl1, agl2, alg13 and alg14 from S.cerevisiae and pglb from C. jejuni that allows the expression of N-glycosylation machinery within the bacteria. Clones were obtained and GALNS activity was evaluated using two different concentrations of inductor (IPTG) as well as two different induction temperatures. From these cultures, it was determined that the best strategy to produce rGALNS was using IPTG 0.1mM and 20 °C. Purified enzymes(with and without N-glycosylations) were used for pH stability assays. The non-glycosylated enzyme showed higher stabilities at physiological pH values, while the N-glycosylated enzyme showed a higher stability at pHs values between 3 and 4, which is similar to that reported for native human GALNS. Kinetics assays were also performed showing a Km and a Vmax of 2.161 mM and 0.1254 U/mL, respectively, for the non-glycosylated enzyme; and 0.504 mM and 0.1583 U/mL, respectively, for the N-glycosylated enzyme. Finally, N-glycosylated GALNS was used in cell capture assays in order to evidence the role of N-glycosylation in the process of internalizing the protein and intracellular traffic. The results showed that N-glycosylated enzyme was taken up by culture cells and that some of the enzyme was targeted to the lysosome. In summary, this study is the first report about the production of andN-glycosylated lysosomal enzyme in E. coli, offering valuable data for its possible usage to the development of an alternative enzyme replacement therapy for Morquio syndrome type A and other lysosomal diseases.