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High‐Level Transient Production of a Heterologous Protein in Plants by Optimizing Induction of a Chemically Inducible Viral Amplicon Expression System
Author(s) -
Plesha Michael A.,
Huang TingKuo,
Dandekar Abhaya M.,
Falk Bryce W.,
McDonald Karen A.
Publication year - 2007
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp070238s
Subject(s) - nicotiana benthamiana , heterologous , agroinfiltration , recombinant dna , microbiology and biotechnology , biology , expression vector , gene expression , heterologous expression , agrobacterium , amplicon , gene , chemistry , biochemistry , transgene , polymerase chain reaction
We have demonstrated that the method of chemical induction using a chemically inducible viral amplicon expression system can be optimized to increase expression of a heterologous protein in plants. A cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce a recombinant human blood protein, α‐1‐antitrypsin (AAT), by co‐infiltrating intact and detached Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Infiltrated plants were induced by either topical applications or pressure injections and inducer was applied at either a single or multiple time points. Applying induction solution every 2 days via topical application resulted in increasing maximum levels of biologically functional rAAT from 0.71% to 1.3% of the total soluble protein (TSP) in detached plant leaves, a 1.8‐fold improvement. Multiple applications of induction solution via pressure injection into intact leaves resulted in maximum levels of biologically functional rAAT being elevated 3‐fold up to 2.4% of TSP compared to 0.8% of TSP when using the conventional method of a single topical application, and expression levels remained high 6 days post‐induction. Overall production of rAAT in intact leaves was found to have a maximum level of 5.8% of TSP or 390 mg rAAT per kg leaf tissue when applying multiple injections of chemical induction solution.