Transient Recombinant Protein Production in Glycoengineered Nicotiana benthamiana Cell Suspension Culture

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 β-1,2-xylosyltransferase and α-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.

[1]  C. Lebrilla,et al.  Purification, characterization, and N‐glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures , 2018, Biotechnology and bioengineering.

[2]  N. Smargiasso,et al.  Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans , 2017, Front. Plant Sci..

[3]  Yoram Tekoah,et al.  Establishment of a tobacco BY2 cell line devoid of plant‐specific xylose and fucose as a platform for the production of biotherapeutic proteins , 2017, Plant biotechnology journal.

[4]  C. Lebrilla,et al.  Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana , 2017, International journal of molecular sciences.

[5]  Florian M. Wurm,et al.  Manufacture of Recombinant Therapeutic Proteins Using Chinese Hamster Ovary Cells in Large‐Scale Bioreactors , 2016 .

[6]  Qiang Chen Glycoengineering of plants yields glycoproteins with polysialylation and other defined N-glycoforms , 2016, Proceedings of the National Academy of Sciences.

[7]  S. Werner,et al.  Engineering of complex protein sialylation in plants , 2016, Proceedings of the National Academy of Sciences.

[8]  M. Sack,et al.  Putting the Spotlight Back on Plant Suspension Cultures , 2016, Front. Plant Sci..

[9]  Yoram Tekoah,et al.  Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience. , 2015, Plant biotechnology journal.

[10]  Sylvain Marcel,et al.  Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals. , 2015, Plant biotechnology journal.

[11]  Friedrich Altmann,et al.  Controlled glycosylation of plant-produced recombinant proteins. , 2014, Current opinion in biotechnology.

[12]  J. Strong,et al.  Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp™ , 2014, Nature.

[13]  D. Bosch,et al.  N-glycosylation of plant-produced recombinant proteins. , 2013, Current pharmaceutical design.

[14]  Jonathan Hurtado,et al.  Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins , 2013, Advanced techniques in biology & medicine.

[15]  토마스 라데마허 Method for the generation and cultivation of a plant cell pack , 2013 .

[16]  W. Curtis,et al.  RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots , 2012, BMC Biotechnology.

[17]  K. McDonald,et al.  Bioreactor systems for in vitro production of foreign proteins using plant cell cultures. , 2012, Biotechnology advances.

[18]  N. Bohorova,et al.  Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant , 2011, Proceedings of the National Academy of Sciences.

[19]  K. McDonald,et al.  Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein , 2011, International journal of molecular sciences.

[20]  M. Jolicoeur,et al.  Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co‐transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing , 2010, Biotechnology progress.

[21]  Archana Belle,et al.  Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax , 2010, Antimicrobial Agents and Chemotherapy.

[22]  L. Vézina,et al.  The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza. , 2010, Plant biotechnology journal.

[23]  S. Leppla,et al.  Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo , 2009, Proceedings of the National Academy of Sciences.

[24]  A. Dandekar,et al.  Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures , 2009, Biotechnology and bioengineering.

[25]  J. Collens,et al.  Agrobacterium‐Mediated Viral Vector‐Amplified Transient Gene Expression in Nicotiana glutinosa Plant Tissue Culture , 2008, Biotechnology progress.

[26]  W. Curtis,et al.  Comparison of Transient Protein Expression in Tobacco Leaves and Plant Suspension Culture , 2008, Biotechnology progress.

[27]  G. Stiegler,et al.  Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. , 2008, Plant biotechnology journal.

[28]  W. Curtis,et al.  Scale‐Up of Agrobacterium‐Mediated Transient Protein Expression in Bioreactor‐Grown Nicotiana glutinosa Plant Cell Suspension Culture , 2008, Biotechnology progress.

[29]  Florian M. Wurm,et al.  Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives , 2007, Biotechnology Letters.

[30]  John A. Young,et al.  Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[32]  Ting-Kuo Huang,et al.  Development of plant cell suspension cultures as a bioproduction platform for recombinant human therapeutic proteins , 2009 .

[33]  J. Collens,et al.  Development of Auxotrophic Agrobacterium tumefaciens for Gene Transfer in Plant Tissue Culture , 2004, Biotechnology progress.