{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T04:44:09Z","timestamp":1776314649043,"version":"3.50.1"},"reference-count":38,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2018,4,16]],"date-time":"2018-04-16T00:00:00Z","timestamp":1523836800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["HDTRA1-15-1-0054"],"award-info":[{"award-number":["HDTRA1-15-1-0054"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["NSF-SSB #1509821"],"award-info":[{"award-number":["NSF-SSB #1509821"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Floyd and Mary Schwall Fellowship in Medical Research","award":["NA"],"award-info":[{"award-number":["NA"]}]},{"name":"Achievement Rewards for College Scientists (ARCS) Scholar Award","award":["NA"],"award-info":[{"award-number":["NA"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 \u03b2-1,2-xylosyltransferase and \u03b1-1,3-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.<\/jats:p>","DOI":"10.3390\/ijms19041205","type":"journal-article","created":{"date-parts":[[2018,4,16]],"date-time":"2018-04-16T12:40:26Z","timestamp":1523882426000},"page":"1205","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["Transient Recombinant Protein Production in Glycoengineered Nicotiana benthamiana Cell Suspension Culture"],"prefix":"10.3390","volume":"19","author":[{"given":"Sara","family":"Sukenik","sequence":"first","affiliation":[{"name":"Department of Chemical Engineering, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kalimuthu","family":"Karuppanan","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3654-0378","authenticated-orcid":false,"given":"Qiongyu","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carlito","family":"Lebrilla","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of California, Davis, CA 95616, USA"},{"name":"Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA"},{"name":"Foods for Health Institute, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Somen","family":"Nandi","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, University of California, Davis, CA 95616, USA"},{"name":"Global HealthShare Initiative, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5145-9968","authenticated-orcid":false,"given":"Karen","family":"McDonald","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, University of California, Davis, CA 95616, USA"},{"name":"Global HealthShare Initiative, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2018,4,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"103","DOI":"10.4172\/2379-1764.1000103","article-title":"Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins","volume":"1","author":"Chen","year":"2013","journal-title":"Adv. Tech. Biol. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1111\/j.1467-7652.2009.00496.x","article-title":"The production of hemagglutinin-based virus-like particles in plants: A rapid, efficient and safe response to pandemic influenza","volume":"8","author":"Couture","year":"2010","journal-title":"Plant Biotechnol. J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1180","DOI":"10.1111\/pbi.12469","article-title":"Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals","volume":"13","author":"Holtz","year":"2015","journal-title":"Plant Biotechnol. J."},{"key":"ref_4","unstructured":"(2018, January 28). Kentucky Bioprocessing Company Website. Available online: https:\/\/www.kentuckybioprocessing.com\/."},{"key":"ref_5","unstructured":"(2018, January 28). Medicago Company Website. Available online: http:\/\/medicago.com\/."},{"key":"ref_6","unstructured":"(2018, January 28). iBio CMO Company Website. Available online: https:\/\/www.ibiocmo.com\/."},{"key":"ref_7","unstructured":"(2018, January 28). Icon Genetics Company Website. Available online: https:\/\/www.icongenetics.com\/."},{"key":"ref_8","unstructured":"(2018, January 28). Fraunhofer Plant-Based Vaccine Factory\u2014Pharmaceutical Technology. Available online: http:\/\/www.pharmaceutical-technology.com\/projects\/fraunhofernewplantba\/."},{"key":"ref_9","unstructured":"Rademacher, T. (2014). Method for the Generation and Cultivation of a Plant Cell Pack. (14\/370,109), U.S. Patent Application."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"398","DOI":"10.1016\/j.biotechadv.2011.07.016","article-title":"Bioreactor systems for in vitro production of foreign proteins using plant cell cultures","volume":"30","author":"Huang","year":"2012","journal-title":"Biotechnol. Adv."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1111\/pbi.12428","article-title":"Large-scale production of pharmaceutical proteins in plant cell culture\u2014The Protalix experience","volume":"13","author":"Tekoah","year":"2015","journal-title":"Plant Biotechnol. J."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"5503","DOI":"10.2174\/1381612811319310006","article-title":"N-glycosylation of plant-produced recombinant proteins","volume":"19","author":"Bosch","year":"2013","journal-title":"Curr. Pharm. Des."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.copbio.2014.06.008","article-title":"Controlled glycosylation of plant-produced recombinant proteins","volume":"30","author":"Strasser","year":"2014","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"9404","DOI":"10.1073\/pnas.1610803113","article-title":"Glycoengineering of plants yields glycoproteins with polysialylation and other defined N-glycoforms","volume":"113","author":"Chen","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1111\/j.1467-7652.2008.00330.x","article-title":"Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure","volume":"6","author":"Strasser","year":"2008","journal-title":"Plant Biotechnol. J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"20690","DOI":"10.1073\/pnas.1108360108","article-title":"Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant","volume":"108","author":"Zeitlin","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1038\/nature13777","article-title":"Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp","volume":"514","author":"Qiu","year":"2014","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5170","DOI":"10.1073\/pnas.0431098100","article-title":"Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor","volume":"100","author":"Scobie","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"12424","DOI":"10.1073\/pnas.0905409106","article-title":"Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo","volume":"106","author":"Liu","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Karuppanan, K., Duhra-Gill, S., Kailemia, M.J., Phu, M.L., Lebrilla, C.B., Dandekar, A.M., Rodriguez, R.L., Nandi, S., and McDonald, K.A. (2017). Expression, purification and biophysical characterization of a secreted anthrax decoy fusion protein in Nicotiana benthamiana. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18010089"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1128\/AAC.00592-10","article-title":"Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax","volume":"55","author":"Wycoff","year":"2011","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"508","DOI":"10.1002\/bit.22061","article-title":"Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures","volume":"102","author":"Huang","year":"2009","journal-title":"Biotechnol. Bioeng."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1021\/bp0703127","article-title":"Scale-up of Agrobacterium-mediated transient protein expression in bioreactor-grown Nicotiana glutinosa plant cell suspension culture","volume":"24","author":"Larsen","year":"2008","journal-title":"Biotechnol. Prog."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"297","DOI":"10.3389\/fpls.2016.00297","article-title":"Putting the spotlight back on plant suspension cultures","volume":"7","author":"Santos","year":"2016","journal-title":"Front. Plant Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1007\/s10529-006-9297-y","article-title":"Recombinant protein production by large-scale transient gene expression in mammalian cells: State of the art and future perspectives","volume":"29","author":"Baldi","year":"2007","journal-title":"Biotechnol. Lett."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Wurm, F.M., and de Jesus, M. (2016). Manufacture of recombinant therapeutic proteins using Chinese hamster ovary cells in large-scale bioreactors. Biosimilars of Monoclonal Antibodies, John Wiley & Sons, Inc.","DOI":"10.1002\/9781118940648.ch11"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"890","DOI":"10.1021\/bp034306w","article-title":"Development of auxotrophic Agrobacterium tumefaciens for gene transfer in plant tissue culture","volume":"20","author":"Collens","year":"2004","journal-title":"Biotechnol. Prog."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"570","DOI":"10.1021\/bp060342u","article-title":"Agrobacterium-mediated viral vector-amplified transient gene expression in Nicotiana glutinosa plant tissue culture","volume":"23","author":"Collens","year":"2007","journal-title":"Biotechnol. Prog."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Larsen, J.S., and Curtis, W.R. (2012). RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots. BMC Biotechnol., 12.","DOI":"10.1186\/1472-6750-12-21"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1021\/bp049569k","article-title":"Comparison of transient protein expression in tobacco leaves and plant suspension culture","volume":"21","author":"Andrews","year":"2005","journal-title":"Biotechnol. Prog."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1534","DOI":"10.1002\/btpr.485","article-title":"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","volume":"26","author":"Boivin","year":"2010","journal-title":"Biotechnol. Prog."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1301","DOI":"10.1002\/bit.26557","article-title":"Purification, characterization and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures","volume":"115","author":"Corbin","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1120","DOI":"10.1111\/pbi.12702","article-title":"Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins","volume":"15","author":"Hanania","year":"2017","journal-title":"Plant Biotechnol. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"403","DOI":"10.3389\/fpls.2017.00403","article-title":"Inactivation of the \u03b2(1,2)-xylosyltransferase and the \u03b1(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 cells by a multiplex CRISPR\/Cas9 strategy results in glycoproteins without plant-specific glycans","volume":"8","author":"Mercx","year":"2017","journal-title":"Front. Plant Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"9498","DOI":"10.1073\/pnas.1604371113","article-title":"Engineering of complex protein sialylation in plants","volume":"113","author":"Kallolimath","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1111\/j.1399-3054.1962.tb08052.x","article-title":"A revised medium for rapid growth and bio assays with tobacco tissue cultures","volume":"15","author":"Murashige","year":"1962","journal-title":"Physiol. Plant."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4975","DOI":"10.3390\/ijms12084975","article-title":"Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein","volume":"12","author":"Arzola","year":"2011","journal-title":"Int. J. Mol. Sci."},{"key":"ref_38","unstructured":"Huang, T.K. (2009). Development of Plant Cell Suspension Cultures as a Bioproduction Platform for Recombinant Human Therapeutic Proteins. [Ph.D. Thesis, University of California]."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/4\/1205\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:00:50Z","timestamp":1760194850000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/4\/1205"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,4,16]]},"references-count":38,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2018,4]]}},"alternative-id":["ijms19041205"],"URL":"https:\/\/doi.org\/10.3390\/ijms19041205","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,4,16]]}}}