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 σs 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 proUmod 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.
Highlights
The leading therapeutic option for MPS IVA is the enzyme replacement therapy (ERT) by using a recombinant enzyme produced in Chinese hamster ovaries (CHO) cells[4]
The staple prokaryotic promoter used in recombinant protein production in E. coli is the lac promoter, which has been widely utilized due to its ability to be straightforwardly controlled with isopropyl β-D-1-thiogalactopyranoside (IPTG) and tightly repressed via lacIq19
Previous studies demonstrated the production of active recombinant human GALNS enzyme (rhGALNS) in E. coli BL21(DE3) using the tac promoter, a synthetic promoter created from the combination of promoters from the trp and lac operons, which is inducible by IPTG21
Summary
The leading therapeutic option for MPS IVA is the enzyme replacement therapy (ERT) by using a recombinant enzyme produced in Chinese hamster ovaries (CHO) cells (elosulfase alfa)[4]. The human GALNS complementary DNA is composed by 1569 bp, encoding a 522 amino acids peptide ( known as precursor peptide) This protein undergoes several posttranslational modifications by the trafficking through the endoplasmic reticulum, Golgi apparatus and lysosome. We explored different approaches to increase the production and activity of rhGALNS in E. coli These approaches included the use of physiologically-regulated promoters to modulate gene expression, induction of osmoprotectants as helpers in protein folding, the overexpression of chaperone proteins, the improvement in the formation of disulfide bonds, and the combination of the different approaches to explore additive effects
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