title: High-level transient production of a heterologous protein in plants by optimizing induction of a chemically inducible viral amplicon expression system.
abstract: 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, alpha-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.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of California at Davis, One Shields Avenue, Davis, California 95616, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Plesha
authors.forename: Ting-Kuo
authors.initials: TK
authors.lastname: Huang
authors.forename: Abhaya M
authors.initials: AM
authors.lastname: Dandekar
authors.forename: Bryce W
authors.initials: BW
authors.lastname: Falk
authors.forename: Karen A
authors.initials: KA
authors.lastname: McDonald
mesh_terms: D017799:Cucumovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D018515:Plant Leaves / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis*
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin