A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was developed that allows for chemically inducible, high-level expression of recombinant proteins in nontransgenic plants. The CMViva expression system was used for production of a heterologous human blood protein, alpha-1-antitrypsin (AAT), in intact and harvested leaves of nontransgenic Nicotiana benthamiana plants using Agrobacterium tumefaciens-mediated transient expression. The post-transcriptional gene silencing suppressor p19 was shown to enhance recombinant AAT expression when used simultaneously with CMViva, and was therefore supplemented during AAT expression. The CMViva expression system allowed for production in intact leaves of up to 390 mg of total recombinant AAT per kg fresh weight of leaf tissue at six days post induction. To facilitate using a production process fully amenable to scale-up, use of harvested plant leaves as well as alternative bioprocessing methods for contacting A. tumefaciens cells and the chemical inducer with the plant cells were investigated. Vacuum application was determined to be the superior method. The duration of vacuum application and concentration of A. tumefaciens cells used were varied to determine their effects on recombinant AAT production. The best bioprocessing conditions for harvested leaves were determined to be a 0.25 minute vacuum infiltration of A. tumefaciens cells with a concentration at an optical density (OD600) of 0.5 (5×108 CFU/mL) along with multiple 1.0 minute vacuum inductions, resulting in production of up to 140 mg total recombinant AAT per kg fresh weight of tissue at six days post induction. The timing between plant leaf harvest and vacuum infiltration was examined, and no differences in transient recombinant AAT production were observed between leaves harvested either 1 hour or 49 hours before infiltration. Expression of p19 at 48 hours before addition of CMViva to express AAT was found not to increase recombinant AAT production over simultaneous addition. Additionally, a mathematical model was developed to predict transient protein expression when using the CMViva expression system. Finally, the bioprocessing techniques used for production of recombinant AAT were transferred for production of an anthrax decoy antitoxin, AntaRx, where production levels up to 1300 mg AntaRx per kg fresh weight of intact leaf were achieved.
|Advisor:||McDonald, Karen A.|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 69/11, Dissertation Abstracts International|
|Keywords:||Biopharmaceuticals, Plant tissues, Recombinant proteins, Viral amplicon|
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