Dataset Information

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 ? 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.

SUBMITTER: Reyes LH

PROVIDER: S-EPMC5517531 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Reyes Luis H LH Cardona Carolina C Pimentel Luisa L Rodríguez-López Alexander A Alméciga-Díaz Carlos J CJ

Scientific reports 20170719 1

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 globa ...[more]

PMID: 28724898

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Project description:BackgroundRecombinant protein production is a process of great industrial interest, with products that range from pharmaceuticals to biofuels. Since high level production of recombinant protein imposes significant stress in the host organism, several methods have been developed over the years to optimize protein production. So far, these trial-and-error techniques have proved laborious and sensitive to process parameters, while there has been no attempt to address the problem by applying Synthetic Biology principles and methods, such as integration of standardized parts in novel synthetic circuits.ResultsWe present a novel self-regulatory protein production system that couples the control of recombinant protein production with a stress-induced, negative feedback mechanism. The synthetic circuit allows the down-regulation of recombinant protein expression through a stress-induced promoter. We used E. coli as the host organism, since it is widely used in recombinant processes. Our results show that the introduction of the self-regulatory circuit increases the soluble/insoluble ratio of recombinant protein at the expense of total protein yield. To further elucidate the dynamics of the system, we developed a computational model that is in agreement with the observed experimental data, and provides insight on the interplay between protein solubility and yield.ConclusionOur work introduces the idea of a self-regulatory circuit for recombinant protein products, and paves the way for processes with reduced external control or monitoring needs. It demonstrates that the library of standard biological parts serves as a valuable resource for initial synthetic blocks that needs to be further refined to be successfully applied in practical problems of biotechnological significance. Finally, the development of a predictive model in conjunction with experimental validation facilitates a better understanding of the underlying dynamics and can be used as a guide to experimental design.

Project description:Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription. Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription.

Dataset Information

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

Publications

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

Similar Datasets

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