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The prospect of economically producing useful biologics in plants has greatly increased with the advent of viral vectors. The ability of viral vectors to amplify transgene expression has seen them develop into robust transient platforms for the high-level, rapid production of recombinant proteins. To adapt these systems to stably transformed plants, new ways of deconstructing the virus machinery and linking its expression and replication to chemically controlled promoters have been developed. The more advanced of these stable, inducible hyper-expression vectors provide both activated and amplified heterologous transgene expression. Such systems could be deployed in broad acre crops and provide a pathway to fully exploit the advantages of plants as a platform for the manufacture of a wide spectrum of products., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.