title: High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.
abstract: Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures.
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, USA.
authors.forename: Ningning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Gonzalez
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, USA.|College of Agriculture and Technology, Arkansas State University, Jonesboro, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Savary
authors.affiliation: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, USA. jxu@astate.edu.|College of Agriculture and Technology, Arkansas State University, Jonesboro, USA. jxu@astate.edu.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Xu
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D049109:Cell Proliferation; D004151:Dipeptides / Q000378:metabolism*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology*
chemical_list: D004151:Dipeptides; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Bioreactor; Hydroxyproline-O-glycosylation; Perfusion culture; Plant cell culture; Recombinant protein