title: Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts.
abstract: Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His₆-MBP). By using the chloroplast 16S rRNA promoter and 5' untranslated region of phage T7 gene 10, GST and His₆-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His₆-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast.
authors.affiliation: Division of Molecular Biosciences, Wolfson Biochemistry Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
authors.forename: Niaz
authors.initials: N
authors.lastname: Ahmad
authors.forename: Franck
authors.initials: F
authors.lastname: Michoux
authors.forename: James
authors.initials: J
authors.lastname: McCarthy
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Nixon
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase