title: Ubiquitin fusion enhances cholera toxin B subunit expression in transgenic plants and the plant-expressed protein binds GM1 receptors more efficiently.
abstract: Developing plant based systems for the production of therapeutic recombinant proteins requires the development of efficient expression strategies and characterization of proteins made in heterologous cellular environment. In this study, the expression of cholera toxin B subunit (CtxB) was examined in the leaves of transgenic tobacco plants. A synthetic gene encoding CtxB was designed for high level expression in plant cells and cloned as ubiquitin (Ub) fusion in a plant expression vector. Tobacco plants were genetically engineered by nuclear transformation to express the CtxB or Ub-CtxB fusion proteins under the control of CaMV35S duplicated enhancer promoter. Functionally active CtxB accumulated in tobacco leaves at 2.5-fold higher level in the Ub-CtxB plants. In the best expressors, CtxB accumulated at 0.9% of the total soluble leaf protein. In both the constructs, molecular mass of the plant-expressed CtxB was 14.6 kDa in contrast to 11.6 kDa for the authentic CtxB. Schiff's test, retention on concanavalin A column and chemical and enzymatic deglycosylation established that the higher molecular mass was due to glycosylation of the CtxB expressed in plant cells. The glycosylated CtxB made in tobacco leaves had higher affinity of binding to the GM1 receptors.
authors.affiliation: National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India.
authors.forename: Satish
authors.initials: S
authors.lastname: Mishra
authors.forename: Dinesh K
authors.initials: DK
authors.lastname: Yadav
authors.forename: Rakesh
authors.initials: R
authors.lastname: Tuli
mesh_terms: D019677:Artificial Gene Fusion; D002772:Cholera Toxin / Q000096:biosynthesis* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006031:Glycosylation; D030821:Plants, Genetically Modified / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism
chemical_list: D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D025801:Ubiquitin; C034993:ganglioside receptor; D002772:Cholera Toxin