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Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is <jats:italic>N<\/jats:italic>-glycosylated upon expression in transgenic <jats:italic>Nicotiana benthamiana<\/jats:italic>. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of <jats:italic>N<\/jats:italic>-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of <jats:italic>PDI<\/jats:italic>, <jats:italic>BiP<\/jats:italic>, <jats:italic>bZIP60<\/jats:italic>, <jats:italic>SKP1<\/jats:italic>, <jats:italic>26S\u03b1<\/jats:italic> proteasome and <jats:italic>PR1a<\/jats:italic> and the detection of ubiquitinated CTB polypeptides revealed that <jats:italic>N<\/jats:italic>-glycosylation significantly relieved ER stress and hypersensitive response and facilitated the folding\/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of <jats:italic>N<\/jats:italic>-glycosylation in increasing recombinant protein yields in plants.<\/jats:p>","DOI":"10.1038\/srep08003","type":"journal-article","created":{"date-parts":[[2015,1,23]],"date-time":"2015-01-23T10:27:58Z","timestamp":1422008878000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":56,"title":["N-Glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential"],"prefix":"10.1038","volume":"5","author":[{"given":"Krystal Teasley","family":"Hamorsky","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J. 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