Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants.
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.
1] Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia. [2].
Benjamin
B
Dugdale
1] Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia. [2].
Cara L
CL
Mortimer
Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia.
Maiko
M
Kato
Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia.
Tess A
TA
James
Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia.
Robert M
RM
Harding
Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia.
James L
JL
Dale
D000431:Ethanol; D017791:Geminiviridae; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D008957:Models, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
D011994:Recombinant Proteins; D000431:Ethanol