Production of recombinant human granulocyte macrophage‐colony stimulating factor in rice cell suspension culture with a human‐like N ‐glycan structure

Summary 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.