Production of Man5GlcNAc2-type sugar chain by the methylotrophic yeast<i>Ogataea minuta</i>

Kuroda, Keiko; Kobayashi, Kazuo; Tsumura, Haruhiko; Komeda, Toshihiro; Chiba, Yasunori; Jigami, Yoshifumi

doi:10.1111/j.1567-1364.2006.00116.x

Cited by 34 publications

(41 citation statements)

References 36 publications

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“…The plasmid pOMEU1, which contains the O. minuta orotidine-5Ј-phosphate decarboxylase (OmURA3) gene and sL/pOMGPA1 and sH/pOMGPU1⌬Sp, which carry the human anti-TRAIL-R antibody light or heavy chain gene fused with a Saccharomyces cerevisiae invertase secretion signal under the control of the OmTDH1 promoter, were prepared as described in our previous studies (23,24). The NdeI-EcoRI-digested plasmid pUC19 (Takara Bio) was subjected to Klenow treatment and was self-ligated.…”

Section: Methodsmentioning

confidence: 99%

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Kuroda

Kobayashi

Kitagawa

et al. 2008

Appl Environ Microbiol

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When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.Antibodies for pharmaceutical applications have recently received a great deal of attention due to their specific antigenbinding activities, antibody-dependent cellular cytotoxicity, and other useful characteristics. Indeed, sales of antibodies for pharmaceuticals have grown rapidly over the past decade and are expected to exceed 30 billion U.S. dollars by 2010 (1). A large volume of antibodies is required for pharmaceuticals, as antibodies are primarily marketed for chronic conditions. Antibodies possess relatively low potency, which results in the need for accumulating doses over time (10). Although therapeutic antibodies have great potential value, they have also been viewed with some skepticism. Antibodies are expensive to produce, in part because they are commonly manufactured by using batch/fed-batch cultures of mammalian cells. The increasing demand to reduce the cost of antibody production has promoted research into the development of an antibody expression system that is more practical than expression with mammalian cells.Development of transgenic plants and animals as alternative hosts is therefore a promising field of study. Monoclonal antibodies have, thus far, been successfully produced from a number of sources, including plants, the milk of transgenic goats, the eggs of transgenic chickens, etc. (4, 12, 32). Moreover, antibodies derived from certain newly developed transgenic systems share physical characteristics that are similar to those of antibodies from mammalian cells such as Chinese hamster ovary (CHO) cells while exhibiting higher antibody-dependent cellular cytotoxicity activity due to the absence of fucose residues in N-linked sugar chains (6, 45). These alternative transgenic expression systems could reduce the cost of large-scale antibody production. However, the prolonged construction of transgenics remains a major disadvantage in terms of market demands.The production of antibodies and antibody fragments has been studied by using various microorganism expression systems, including Escherichia coli, fungus, and yeast (7,13,37,41). Since microorg...

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Section: Methodsmentioning

confidence: 99%

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Kuroda

Kobayashi

Kitagawa

et al. 2008

Appl Environ Microbiol

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“…In this study, we used a methylotrophic yeast, Ogataea minuta, in which the OCH1 gene is disrupted (21) and the Mnn4p (MNN4) gene is overexpressed (22,23), as host cells to produce a recombinant GLA having mammalianlike, phosphorylated N-glycans, and examined its effects on the organs of Fabry mice.…”

Section: Efficient Uptake Of Recombinant α-Galactosidase a Produced Wmentioning

confidence: 99%

“…The polymerase chain reaction fragment obtained was digested with XbaI and BamHI, and the resultant DNA fragment was inserted between the XbaI and BamHI sites of the pOMEU1 vector (21). On the other hand, plasmid pOMEG-ScMNN4 (25) was used for overproduction of ScMnn4p in O. minuta.…”

Section: Plasmid Construction and Transformation Of A Methylotrophic mentioning

confidence: 99%

“…On the other hand, plasmid pOMEG-ScMNN4 (25) was used for overproduction of ScMnn4p in O. minuta. These vectors were cut with NotI and used for transformation of the O. minuta TK-3-A strain (Δoch1) (21), which was provided by Kyowa Hakko Kirin (Tokyo, Japan). Transformation of O. minuta was performed as described previously (21,25).…”

Section: Plasmid Construction and Transformation Of A Methylotrophic mentioning

confidence: 99%

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Efficient Uptake of Recombinant α-Galactosidase A Produced with a Gene-Manipulated Yeast by Fabry Mice Kidneys

Tsukimura

Kawashima

Togawa

et al. 2011

Mol Med

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To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and overexpressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.

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“…Since high expression of recombinant enzyme was expected, we chose the methylotrophic yeast O. minuta as a host for recombinant HexA expression. As with S. cerevisiae, an OCH1 disrupted strain, O. minuta TK5-3, 103) was used as a host. The genes coding for the a and b subunits of HexA were co-expressed in O. minuta TK5-3 under the alcohol oxidase (AOX1) promoter.…”

Section: Treatment Of Lysosomal Storage Diseasesmentioning

confidence: 99%

Glycan Engineering and Production of 'Humanized' Glycoprotein in Yeast Cells

Chiba

Akeboshi

2009

Biological & Pharmaceutical Bulletin

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Protein therapeutics, such as antibodies and cytokines, is the largest class of new drug candidates being developed by pharmaceutical companies. Although most of these glycoproteins are produced in mammalian cells, there is concern that their large-scale production could be affected by an inadequate supply of bovine serum. There is also the risk of viral infection spreading through the use of contaminated protein therapeutics. Consequently, protein expression systems in yeast have been established because protein manufacturing costs are cheaper than in mammalian cells, and yeast systems are virus-free. However, yeasts cannot generate human-type glycans, and thus cannot produce therapeutic glycoproteins for human use. There has therefore been considerable interest in glycan remodeling, from yeast-type to human-type. 'Humanized' glycoproteins can now be generated in yeast by disrupting yeast-specific glycosyltransferases and introducing genes responsible for sugar-nucleotide synthesis, its transported from the cytosol to Golgi lumen, as well as their transfer and hydrolysis. A compound that inhibits yeast O-mannosyltransferase suppresses yeast-specific O-mannosyl modification, and can produce mucin-type glycoproteins. These systems are just being developed to the stage where the production in glycoengineered yeast of biopharmaceutical glycoproteins such as cytokines, antibodies for therapeutics, and enzymes for replacement therapy for lysosomal diseases are being evaluated for clinical applications. Yeast glycoprotein expression systems are expected to become the dominant approach for the production of human glycoproteins in the near future.

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Production of Man5GlcNAc2-type sugar chain by the methylotrophic yeastOgataea minuta

Cited by 34 publications

References 36 publications

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Efficient Uptake of Recombinant α-Galactosidase A Produced with a Gene-Manipulated Yeast by Fabry Mice Kidneys

Glycan Engineering and Production of 'Humanized' Glycoprotein in Yeast Cells

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