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Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants

Nature Protocols volume 9, pages 1010–1027 (2014)Cite this article

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Abstract

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6–9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

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Figure 1: Schematic representation of the INPACT-inducible, hyperexpression platform.
Figure 2: Vectors generated for the optimization and construction of the INPACT-GOI cassette.
Figure 3: Agroinfiltration of N. tabacum leaves for transient gene expression.
Figure 4: INPACT-directed expression of human vitronectin in transgenic tobacco.

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Acknowledgements

We thank the Australian Research Council for funding this research.

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Author notes

  1. Benjamin Dugdale and Cara L Mortimer: These authors jointly directed this work.

Authors and Affiliations

  1. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia

    Benjamin Dugdale, Cara L Mortimer, Maiko Kato, Tess A James, Robert M Harding & James L Dale

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  1. Benjamin Dugdale
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Contributions

B.D. and C.L.M. designed and performed experiments, analyzed data and wrote the manuscript. M.K. and T.A.J. designed and performed experiments and analyzed data. J.L.D. and R.M.H. designed experiments, analyzed data and wrote the manuscript.

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Correspondence to James L Dale.

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Syntron and LIR sequence (PDF 96 kb)

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Dugdale, B., Mortimer, C., Kato, M. et al. Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants. Nat Protoc 9, 1010–1027 (2014). https://doi.org/10.1038/nprot.2014.068

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