title: In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants.
abstract: In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the β-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.
authors.affiliation: Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane 4000, Queensland, Australia.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dugdale
authors.forename: Cara L
authors.initials: CL
authors.lastname: Mortimer
authors.forename: Maiko
authors.initials: M
authors.lastname: Kato
authors.forename: Tess A
authors.initials: TA
authors.lastname: James
authors.forename: Robert M
authors.initials: RM
authors.lastname: Harding
authors.forename: James L
authors.initials: JL
authors.lastname: Dale
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D002417:Cattle; D017796:Caulimovirus / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology; D017791:Geminiviridae / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000821:virology; D019076:Transgenes / Q000235:genetics*; D014362:Trypsinogen / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics; D019096:Vitronectin / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D019096:Vitronectin; D000431:Ethanol; D014362:Trypsinogen; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D005966:Glucuronidase