Dataset Information

Glycosylation and functionality of recombinant ?-glucocerebrosidase from various production systems.

ABSTRACT: The glycosylation of recombinant ?-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize ?-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different cell systems and undergo various post-translational or post-production glycosylation modifications to expose their mannose residues. This is the first study in which the glycosylation profiles of the three enzymes are compared, using the same methodology and the effect on functionality and cellular uptake is evaluated. While the major differences in glycosylation profiles reside in the variation of terminal residues and mannose chain length, the enzymatic activity and stability are not affected by these differences. Furthermore, the cellular uptake and in-cell stability in rat and human macrophages are similar. Finally, in vivo studies to evaluate the uptake into target organs also show similar results for all three enzymes. These results indicate that the variations of glycosylation between the three regulatory-approved ?-glucocerebrosidase enzymes have no effect on their function or distribution.

SUBMITTER: Tekoah Y

PROVIDER: S-EPMC3782720 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems.

Tekoah Yoram Y Tzaban Salit S Kizhner Tali T Hainrichson Mariana M Gantman Anna A Golembo Myriam M Aviezer David D Shaaltiel Yoseph Y

Bioscience reports 20130925 5

The glycosylation of recombinant β-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize β-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different ce ...[more]

PMID: 23980545

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Project description:BackgroundThe microbes Escherichia coli and Pichia pastoris are convenient prokaryotic and eukaryotic hosts, respectively, for the recombinant production of proteins at laboratory scales. A comparative study was performed to evaluate a range of constructs and process parameters for the heterologous intra- and extracellular expression of genes encoding the industrially relevant enzyme galactose 6-oxidase (EC 1.1.3.9) from the fungus Fusarium graminearum. In particular, the wild-type galox gene from F. graminearum, an optimized variant for E. coli and a codon-optimized gene for P. pastoris were expressed without the native pro-sequence, but with a His-tag either at the N- or the C-terminus of the enzyme.ResultsThe intracellular expression of a codon-optimized gene with an N-terminal His10-tag in E. coli, using the pET16b+ vector and BL21DE3 cells, resulted in a volumetric productivity of 180 U·L-1·h-1. The intracellular expression of the wild-type gene from F. graminearum, using the pPIC3.5 vector and the P. pastoris strain GS115, was poor, resulting in a volumetric productivity of 120 U·L-1·h-1. Furthermore, this system did not tolerate an N-terminal His10-tag, thus rendering isolation of the enzyme from the complicated mixture difficult. The highest volumetric productivity (610 U·L-1·h-1) was achieved when the wild-type gene from F. graminearum was expressed extracellularly in the P. pastoris strain SMD1168H using the pPICZ?-system. A C-terminal His6-tag did not significantly affect the production of the enzyme, thus enabling simple purification by immobilized metal ion affinity chromatography. Notably, codon-optimisation of the galox gene for expression in P. pastoris did not result in a higher product yield (g protein·L-1 culture). Effective activation of the enzyme to generate the active-site radical copper complex could be equally well achieved by addition of CuSO4 directly in the culture medium or post-harvest.ConclusionsThe results indicate that intracellular production in E. coli and extracellular production in P. pastoris comprise a complementary pair of systems for the production of GalOx. The prokaryotic host is favored for high-throughput screening, for example in the development of improved enzymes, while the yeast system is ideal for production scale-up for enzyme applications.

Dataset Information

Glycosylation and functionality of recombinant ?-glucocerebrosidase from various production systems.

Publications

Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems.

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