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1. (Article ID: 8666)
 
Piirainen MA, Boer H, Ruijter JC, Frey AD
A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae
Glycoconjugate Journal 33(2) (2016) 189-199
 

N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae.

glycoengineering, N-glycosylation, glycosylation efficiency, yeast, MNN1, UDP-GlcNAc transporter

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2. (Article ID: 8716)
 
Sakaki Y, Tashiro M, Katou M, Sakuma C, Hirano T, Hakamata W, Nishio T
Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine and melibiose using Aspergillus niger α-galactosidase, and properties of the products
Bioscience, Biotechnology, and Biochemistry 80(9) (2016) 1836-1842
 

Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf.

oligosaccharide, transgalactosylation, N-acetylplateosamine, N-acetylraffinosamine, α-galactosidase

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