Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 19, Issue 4, Pages -Publisher
MDPI
DOI: 10.3390/ijms19041205
Keywords
Agrobacterium tumefaciens; plant cell culture; co-culture; recombinant protein expression; N-glycosylation
Funding
- Defense Threat Reduction Agency [HDTRA1-15-1-0054]
- National Science Foundation (NSF-SSB) [1509821]
- Floyd and Mary Schwall Fellowship in Medical Research
- Achievement Rewards for College Scientists (ARCS) Scholar Award
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1509821] Funding Source: National Science Foundation
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Transient recombinant protein production is a promising alternative to stable transgenic systems, particularly for emergency situations in which rapid production of novel therapeutics is needed. In plants, Agrobacterium tumefaciens can be used as a gene delivery vector for transient expression. A potential barrier for plant-based production of human therapeutics is that different glycosylation patterns are found on plant and mammalian proteins. Since glycosylation can affect the efficacy, safety and stability of a therapeutic protein, methods to control glycan structures and distributions in plant-based systems would be beneficial. In these studies, we performed Agrobacterium-mediated transient expression in glycoengineered plant cell suspension cultures. To reduce the presence of plant-specific glycans on the product, we generated and characterized cell suspension cultures from beta-1,2-xylosyltransferase and alpha-1,3-fucosyltransferase knockdown Nicotiana benthamiana. An anthrax decoy fusion protein was transiently produced in these glycoengineered plant cell suspension cultures through co-culture with genetically engineered Agrobacterium. The mass ratio of Agrobacterium to plant cells used was shown to impact recombinant protein expression levels. N-glycosylation analysis on the anthrax decoy fusion protein produced in glycoengineered N. benthamiana showed a dramatic reduction in plant-specific N-glycans. Overall, the results presented here demonstrate the feasibility of a simple, rapid and scalable process for transient production of recombinant proteins without plant-specific glycans in a glycoengineered plant cell culture host.
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