title: Rapid system for evaluating bioproduction capacity of complex pharmaceutical proteins in plants.
abstract: Transgene product yield remains a key limitation in commercializing plant-derived pharmaceutical proteins. Although significant progress has been made in understanding the roles of promoters, enhancers, integration sites, codon usage, cryptic RNA sites, silencing, and product compartmentalization on product yield and quality, researchers still cannot reliably predict which proteins will be produced at high levels or what manipulations will guarantee enhanced productivity. We have optimized a simple transient expression system in Nicotiana benthamiana enabling rapid assessment of transgene potential for plant-based bioproduction. Briefly, intact Nicotiana benthamiana plants are vacuum-infiltrated with Agrobacterium tumefaciens cultures carrying the transgene of interest. After 48-96 h of further incubation, leaves are harvested for protein characterization. Using the immunomodulator interleukin-12 as a model pharmaceutical protein, we obtained bioactive recombinant protein at levels exceeding 5% of total soluble leaf protein. Appropriately assembled multimeric proteins have also been obtained following coinfiltration with Agrobacterium tumefaciens strains individually encoding each subunit. This system provides a rapid source of transgene product for assessing posttranslational modifications, purification strategies, and bioactivity as well as an effective system for optimizing construct elements. For vaccines, product purified from two to eight plants may support mouse vaccination trials providing efficacy and immune assessment data early in the development process.
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Medrano
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Reidy
authors.forename: Jianyun
authors.initials: J
authors.lastname: Liu
authors.forename: Jorge
authors.initials: J
authors.lastname: Ayala
authors.forename: Maureen C
authors.initials: MC
authors.lastname: Dolan
authors.forename: Carole L
authors.initials: CL
authors.lastname: Cramer
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins / Q000096:biosynthesis; D014026:Nicotiana / Q000235:genetics*
chemical_list: D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins