期刊
JOURNAL OF BIOTECHNOLOGY
卷 143, 期 2, 页码 95-102出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2009.06.016
关键词
OCH1; MNN1; N-Glycosylation; Mannosyltransferases; Glycoproteins; Kluyveromyces lactis
资金
- 863 High-tech Project of China Ministry of Science and Technology [2007AA02Z103]
- GeneScan 3.7 software [ABI 3100 DNA]
Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding alpha 1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative alpha 1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The Winked oligosaccharides assembled on a secretory glycoprotein, HSA/GM-CSF in Kloch1 mutant, contained oligosaccharide Man(13-14)GlcNAc(2), and in Kloch1 mnn1 mutant, contained oligosaccharide Man(9-11)GlcNAc(2), whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man(>30)GlcNAc(2). Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of Winked oligosaccharides in K lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation. most possibly encodes alpha 1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production. (C) 2009 Elsevier B.V. All rights reserved.
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