title: Production of recombinant human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure.
abstract: The rice α-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous α-1,3-fucosyltransferase (α-1,3-FucT) and β-1,2-xylosyltransferase (β-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of α-1,3-fucosyltransferase and β-1,2-xylosyltransferase (Δ3FT/XT)-9 glyco-engineered line with significantly reduced core α-1,3-fucosylated and/or β-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific α-1,3-fucose and/or β-1,2-xylose residues incorporated into recombinant human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + Δ3FT/XT-4 glyco-engineered line co-expressing ihpRNA of Δ3FT/XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium.
authors.affiliation: Jeonju Biomaterials Institute, Jang-dong, Jeonju, South Korea.
authors.forename: Yun-Ji
authors.initials: YJ
authors.lastname: Shin
authors.forename: Yun-Jo
authors.initials: YJ
authors.lastname: Chong
authors.forename: Moon-Sik
authors.initials: MS
authors.lastname: Yang
authors.forename: Tae-Ho
authors.initials: TH
authors.lastname: Kwon
mesh_terms: D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D005643:Fucose / Q000378:metabolism; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014994:Xylose / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D005643:Fucose; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D014994:Xylose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D010430:Pentosyltransferases; C408586:beta 1,2-xylosyltransferase