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

Dugdale, Benjamin, Mortimer, Cara L., Kato, Maiko, James, Tess A., Harding, Robert M., & Dale, James L. (2014) Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants. Nature Protocols, 9(5), pp. 1010-1027.

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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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ID Code: 70642
Item Type: Journal Article
Refereed: Yes
Additional URLs:
DOI: 10.1038/nprot.2014.068
ISSN: 1750-2799 (online) 1754-2189 (print)
Divisions: Current > Research Centres > Centre for Tropical Crops and Biocommodities
Past > QUT Faculties & Divisions > Faculty of Science and Technology
Current > Institutes > Institute for Future Environments
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2014 Nature America, Inc. All rights reserved.
Deposited On: 30 Apr 2014 01:01
Last Modified: 30 Apr 2014 01:01

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