High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector

Abstract

We describe here a unique ethanol-inducible process for expression of recombinant proteins in transgenic plants. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the uninduced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. Transgenic Nicotiana benthamiana plants incorporating this double-inducible system demonstrate negligible background expression, high (over 0.5 × 10 4 -fold) induction multiples, and high absolute levels of protein expression upon induction (up to 4.3 mg∕g fresh biomass). The process can be easily scaled up, supports expression of practically important recombinant proteins, and thus can be directly used for industrial manufacturing.plant-made pharmaceuticals | TMV

Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins

“…The methods used to construct and apply TMV-and PVXbased magnICON vectors and TMV-based vectors for EtOH-inducible expression (Figs. S1 and S3) were as described previously (11,12,15). Assembly of the binary construct for EtOH-inducible expression was done by modular cloning as described in ref.…”

Type III-Dependent Translocation of HrpB2 by a Nonpathogenic hpaABC Mutant of the Plant-Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria

“…vesicatoria strains were inoculated into leaves of the near-isogenic pepper cultivars Early Cal Wonder (ECW), ECW-10R, and ECW-30R, as well as into leaves of gfp (green fluorescent protein)-transgenic N. benthamiana at a concentration of 4 ϫ 10 8 CFU ml Ϫ1 in 1 mM MgCl 2 if not stated otherwise (30,48,49). gfp-transgenic N. benthamiana plants were generated using the viral construct pICH18951, which contains the gfp gene and the RNA-dependent RNA polymerase (RdRp)-encoding sequence downstream of the alcA promoter as described previously (50). After infection, pepper plants were incubated in an incubation chamber for 16 h of light at 28°C and 65% humidity and 8 h of darkness at 22°C and 65% humidity.…”

“…The amplification of the viral vector in the presence of dTALE-2 results in a high-level expression of gfp. The resulting GFP fluorescence is locally restricted to infected cells because the viral vector lacks coding sequences for the movement and coat proteins, which are needed for cell-to-cell and systemic movement (50,63).…”

“…dTALE-2 is a derivative of the TAL effector AvrBs3 and contains modified RVDs, which were designed to bind to the DNA target sequence 5=-TCCCCGCATAG CTGAACAT-3= (63). As a reporter system, we used transgenic N. benthamiana plants containing a stably integrated tobacco mosaic virus (TMV)-based viral vector which encodes an RdRp and GFP (50,63). The viral vector construct was placed under the control of the alcA promoter from Aspergillus nidulans, which requires binding of the AlcR transcriptional activator for transcription activation.…”

In Plant Activation: An Inducible, Hyperexpression Platform for Recombinant Protein Production in Plants

“…Perhaps the only system to fulfill these criteria was recently reported by Werner et al (2011), who adapted the transient TMV-based replicon vector system to transgenic plants by linking its expression to the ethanol-responsive AlcA:AlcR gene switch (Caddick et al, 1998). In the absence of the ethanol inducer molecule, N. benthamiana plants displayed negligible reporter gene expression; however, upon induction, the transgene was amplified and expressed from viral RNA replicons and plants accumulated high absolute levels of recombinant protein comparable to those observed transiently.…”