Improvement in the production of the human recombinant enzyme N-acetylgalactosamine-6-sulfatase (rhGALNS) in Escherichia coli using synthetic biology approaches

Abstract

Previously, we demonstrated production of an active recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS) enzyme in Escherichia coli as a potential therapeutic alternative for mucopolysaccharidosis IVA. However, most of the rhGALNS produced was present as protein aggregates. Here, several methods were investigated to improve production and activity of rhGALNS. These methods involved the use of physiologically-regulated promoters and alternatives to improve protein folding including global stress responses (osmotic shock), overexpression of native chaperones, and enhancement of cytoplasmic disulfide bond formation. Increase of rhGALNS activity was obtained when a promoter regulated under σ s was implemented. Additionally, improvements were observed when osmotic shock was applied. Noteworthy, overexpression of chaperones did not have any effect on rhGALNS activity, suggesting that the effect of osmotic shock was probably due to a general stress response and not to the action of an individual chaperone. Finally, it was observed that high concentrations of sucrose in conjunction with the physiological-regulated promoter proU mod significantly increased the rhGALNS production and activity. Together, these results describe advances in the current knowledge on the production of human recombinant enzymes in a prokaryotic system such as E. coli, and could have a significant impact on the development of enzyme replacement therapies for lysosomal storage diseases.

Transcriptional Analyses of Acute Exposure to Methylmercury on Erythrocytes of Loggerhead Sea Turtle

“…We identified the following upregulated genes: HEX_A, which catalyzes the degradation of GM2 gangliosides to GM3 gangliosides in lysosomes [90], molecules which are found in the plasma membrane [91]; MANB, a glycosyl hydrolase that degrades polysaccharides [92]; GALNS, another lysosomal hydrolase that degrades proteins such as glycosaminoglycans, keratan sulfate, and chondroitin-6-sulfate [93]; and AP4B1, which is involved in the generation of vesicles and, in the charge selection, controls the vesicular transport of proteins in different traffic pathways and contributes to the spatial control of autophagy [94]. Apparently, the erythrocyte membrane generates lipids and carbohydrates as a result of ROS-triggered events, such as lipid peroxidation.…”

Metabolic engineering strategies for butanol production in Escherichia coli

“…Moreover, E. coli possesses several industrially relevant characteristics like (a) capacity to grow on mineral media, (b) utilization of different substrates as carbon source, (c) fast growth rate, (d) ability to grow under aerobic and anaerobic conditions, (e) robustness under industrial conditions, and (f) tolerance to high concentrations of substrates and products (Clomburg & Gonzalez, 2010;Koppolu & Vasigala, 2016). Hence, E. coli has been successfully engineered for the production of diverse products, including hormones (Rezaei & Zarkesh-Esfahani, 2012), proteins (Reyes, Cardona, Pimentel, Rodríguez-López, & Alméciga-Díaz, 2017), and amino acids . Particularly, its potential to produce biofuels was first explored with ethanol, a native product (Ohta, Beall, Mejia, Shanmugam, & Ingram, 1991).…”

Research, diagnosis and education in inborn errors of metabolism in Colombia: 20 years’ experience from a reference center

“…For ERT we have reported the production and characterization of human recombinant lysosomal iduronate-2-sulfate sulfatase (IDS), N-acetylgalactosamine-6-sulfate sulfatase (GALNS), and β-N-acetylhexosaminidases (Hex-A, Hex-B, and Hex-S) in the bacteria Escherichia coli and the yeast Pichia pastoris [ 68 – 74 ], as well as the phenylalanine hydroxylase in Lactobacillus plantarum [ 75 ] (Table 4 ). In the two first expression platforms, we have evaluated different strains, vectors, and culture conditions [ 73 , 76 – 80 ]. All recombinant proteins have shown activity levels similar or higher that those reported for native or recombinant proteins produced in other expression systems, even IDS and GALNS produced in E. coli [ 73 , 81 ].…”