title: Efficient plant-based production of chicken interleukin-12 yields a strong immunostimulatory cytokine.
abstract: Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic in mammals. Therapeutic strategies to develop mammalian IL-12 as a vaccine adjuvant/immunomodulator for promoting cellular immunity and establishing a Th1-biased immune response further support the potential value of ChIL-12. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. We have expressed, characterized, and purified biologically active ChIL-12 in plants using a rapid Agrobacterium-mediated tobacco plant-based transient expression system. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of chicken IL-12 (ChIL-12). A histidine 6x tag was used for identity and purification of ChIL-12(His) protein. Our results demonstrated precise cleavage of the endogenous chicken p40 signal peptide in plants as well as addition of N-linked glycans. Biological activity was confirmed in vitro by interferon-gamma secretion of ChIL-12-treated chicken splenocytes. In addition, splenocytes treated with ChIL-12 expressed with or without the His tag demonstrated comparable ChIFN-gamma induction. These studies indicate that plant-based platforms for bioproduction of complex pharmaceutical proteins produce functional ChIL-12 and provide key advantages in safety, scale, and cost-effective platform for veterinary vaccine and therapeutic applications.
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, Arkansas 72467, USA.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Medrano
authors.forename: Maureen C
authors.initials: MC
authors.lastname: Dolan
authors.forename: Nathan T
authors.initials: NT
authors.lastname: Stephens
authors.forename: Anthony
authors.initials: A
authors.lastname: McMickle
authors.forename: Gisela
authors.initials: G
authors.lastname: Erf
authors.forename: David
authors.initials: D
authors.lastname: Radin
authors.forename: Carole L
authors.initials: CL
authors.lastname: Cramer
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens / Q000235:genetics*; D006031:Glycosylation; D018664:Interleukin-12 / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D018515:Plant Leaves / Q000235:genetics; D055550:Protein Stability; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D018664:Interleukin-12