Zhang Ningning, Gonzalez Maria, Savary Brett, Xu Jianfeng
Arkansas Biosciences Institute, Arkansas State University, Jonesboro, USA.
College of Agriculture and Technology, Arkansas State University, Jonesboro, USA.
Biotechnol J. 2016 Mar;11(4):497-506. doi: 10.1002/biot.201500377. Epub 2015 Dec 30.
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.
低产量蛋白质生产仍然是植物细胞培养技术最显著的经济障碍。重组蛋白与羟脯氨酸-O-糖基化设计糖肽标签的融合一直能提高分泌蛋白产量。这促使我们研究该技术的工艺开发，旨在实现商业可行性所需的生产力水平。我们使用表达绿色荧光蛋白（EGFP）并与由32个“Ser-Pro”二肽重复序列组成的糖肽标签（即(SP)32）融合的烟草BY-2细胞培养物，来研究细胞生长和蛋白质分泌、扩大培养规模以及建立用于连续生产的灌注培养。BY-2细胞在 Schenk & Hildebrandt 培养基中积累的细胞生物量水平较低（约7.5 g干重/L），但分泌的(SP)32标记的EGFP产量很高（125 mg/L）。该细胞培养物的蛋白质生产力在6.0年内一直保持稳定。在5-L生物反应器中培养的BY-2细胞同样产生了131 mg/L的高分泌蛋白产量。在0.25和0.5天-1的灌注速率下，细胞灌注培养成功运行了30天，分别产生了17.6和28.9 mg/天/L的蛋白质体积生产力。这项研究证明了设计糖肽技术在利用植物细胞培养商业化生产有价值蛋白质方面的巨大潜力。
