Glycosylation of Recombinant Proteins in Plan

Gorr, Gilbert; Altmann, Friedrich

doi:10.1002/9783527619771.ch15

Cited by 4 publications

(5 citation statements)

References 160 publications

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“…The presence of large amounts of Lewis a determinants on rhEPO is in clear contrast with other proteins expressed in Physcomitrella . On antibodies, this structure was found as a minor fraction, if present at all (Gorr and Altmann, 2006). One explanation of the different complexity of the N ‐glycan pattern of different proteins may be that glycosylation is not only dependent on the host, but also influenced by the three‐dimensional structure of the protein itself, causing differential accessibility of potential N ‐glycosylation sites.…”

Section: Discussionmentioning

confidence: 99%

High‐level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Δ‐fuc‐t Δ‐xyl‐t mutant

Weise¹,

Altmann

Rodriguez‐Franco

et al. 2007

Plant Biotechnology Journal

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107

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SummaryThe highly glycosylated peptide hormone erythropoietin (EPO) plays a key role in the regulation of erythrocyte maturation. Currently, marketed EPO is produced by recombinant technology in mammalian cell cultures. The complementary DNA (cDNA) for human EPO (hEPO) was transiently and stably expressed in the moss Physcomitrella patens wild-type and ∆ -fuc-t ∆ -xyl-t mutant, the latter containing N -glycans lacking the plant-specific, corebound α 1,3-fucose and β 1,2-xylose. New expression vectors were designed based on a Physcomitrella ubiquitin gene-derived promoter for the expression of hEPO cDNA. Transient expression in protoplasts was much stronger at 10 than at 20 ° C. In Western blot analysis, the molecular size of moss-produced recombinant human EPO (rhEPO) was identified to be 30 kDa, and it accumulated in the medium of transiently transformed protoplasts to high levels around 0.5 µ g/mL. Transgenic Physcomitrella ∆ -fuc-t ∆ -xyl-t mutant lines expressing EPO cDNA showed secretion of rhEPO through the cell wall to the culture medium. In 5-and 10-L photobioreactor cultures, secreted rhEPO accumulated to high levels above 250 µ g/g dry weight of moss material after 6 days. Silver staining of rhEPO on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) taken from the bioreactor culture demonstrated a high purity of the over-expressed secreted rhEPO, with a very low background of endogenous moss proteins. Peptide mapping of rhEPO produced by the Physcomitrella ∆ -fuc-t ∆ -xyl-t mutant indicated correct processing of the plant-derived signal peptide. All three N -glycosylation sites of rhEPO were occupied by complex-type N -glycans completely devoid of the plant-specific core sugar residues fucose and xylose.

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Section: Discussionmentioning

confidence: 99%

High‐level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Δ‐fuc‐t Δ‐xyl‐t mutant

Weise¹,

Altmann

Rodriguez‐Franco

et al. 2007

Plant Biotechnology Journal

Self Cite

107

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“…The two larger rFH bands could clearly be detected in the elution fractions of line FH3 but not in WT fractions. As alpha1,3 fucosylation occurs late in the maturation of complex type but not on premature oligomannosidic glycans (Gorr and Altmann, 2006), we assume that the observed size differences in the larger bands resulted from a different grade of occupation of the glycosylation sites rather than from incomplete processing of the attached glycans. As it was suggested that the roles of FH N‐glycans may be structural rather than functional (Fenaille et al.…”

Section: Resultsmentioning

confidence: 92%

“…However, to avoid immune reactions against these residues (Bencurova et al. , 2004; Gorr and Altmann, 2006) which may occur when a recombinant protein with native plant glycosylation would be applied to patients, the rFH will have to be expressed in a glyco‐engineered moss production strain.…”

Section: Resultsmentioning

confidence: 99%

Production of biologically active recombinant human factor H in Physcomitrella

Büttner-Mainik

Parsons

Jerome

et al. 2010

Plant Biotechnology Journal

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SummaryThe human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry.Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma.

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“…Moss knockout strains were created which lacked the enzymes responsible for xylosylation and fucosylation, beta 1,2-xylosyltransferase and alpha1,3-fucosyltransferase, respectively [58]. The resulting double knockout strains were shown to be completely devoid of the allergenic sugar residues and were employed to synthesize several products of pharmaceutical value, including human VEGF [58], antibodies IgG1 [59] and IgG4 [60], and erythropoietin [61]. For all products analysed so far, over-glycosylation never was detected (G. Gorr, personal communication).…”

Section: Genetic Engineering Of Moss Strains For Enhanced Product Yieldsmentioning

confidence: 99%

“…However, antibodies, comprising the probably most interesting and largest group of biotech drugs in use and clinical development [69] have one conserved glycosylation site and show sialylation of only a small fraction of the attached N-glycan. Moreover, CHO cells, standard system for production of therapeutical antibodies, tend to undersialylate the recombinant protein [61]. Therefore, plant-based antibody production with appropriate glycosylation is a realizable task, which was demonstrated for moss recently [60].…”

Section: Genetic Engineering Of Moss Strains For Enhanced Product Yieldsmentioning

confidence: 99%

Current achievements in the production of complex biopharmaceuticals with moss bioreactors

Decker

Reski

2007

Bioprocess Biosyst Eng

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Transgenic plants are promising alternatives for the low-cost and safe pathogen-free production of complex recombinant pharmaceutical proteins (molecular farming). Plants as higher eukaryotes perform posttranslational modifications similar to those of mammalian cells. However, plant-specific protein N-glycosylation was shown to be immunogenic, a fact that represents a drawback for many plant systems in biopharmaceutical production. The moss Physcomitrella patens offers unique properties as a contained system for protein production. It is grown in the predominant haploid gametophytic stage as tissue suspension cultures in photobioreactors. Efficient secretory signals and a transient transfection system allow the secretion of freshly synthesized proteins to the surrounding medium. The key advantage of Physcomitrella compared to other plant systems is the feasibility of targeted gene replacements. By this means, moss strains with non-immunogenic humanized glycan patterns were created. Here we present an overview of the relevant aspects for establishing moss as a production system for recombinant biopharmaceuticals.

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Glycosylation of Recombinant Proteins in Plan

Cited by 4 publications

References 160 publications

High‐level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Δ‐fuc‐t Δ‐xyl‐t mutant

High‐level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Δ‐fuc‐t Δ‐xyl‐t mutant

Production of biologically active recombinant human factor H in Physcomitrella

Current achievements in the production of complex biopharmaceuticals with moss bioreactors

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