High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.
High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.
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使用设计糖肽标签在烟草细胞培养物中表达重组蛋白的高产量分泌：工艺开发。
DOI：
10.1002/biot.201500377
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发表时间：
2016
影响因子：
4.7
通讯作者：
Xu,Jianfeng
中科院分区：
文献类型：
--
作者：
Zhang,Ningning;Gonzalez,Maria;Savary,Brett;Xu,Jianfeng
低产量蛋白质生产仍然是植物细胞培养技术最重要的经济障碍。重组蛋白与羟脯氨酸-O-糖基化设计糖肽标签的融合一贯地提高了分泌蛋白的产量。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EGFP的烟草BY-2细胞培养物作为与由32个重复的“Ser-Pro”二肽或（SP）32组成的糖肽标签的融合物，以研究细胞生长和蛋白质分泌、培养物放大以及用于连续生产的灌注培养物的建立。BY-2细胞在Schenk & Hildebrandt培养基中积累低水平的细胞生物量（约7.5 g DW/L），但分泌高产率的（SP）32标记的EGFP（125 mg/L）。细胞培养物的蛋白质生产率已稳定6.0年。在5 L生物反应器中培养的BY-2细胞类似地产生131 mg/L的高分泌蛋白产量。在0.25和0.5天-1的灌流率下成功地进行了30天的细胞灌流培养，分别产生了17.6和28.9 mg/天/L的蛋白质体积生产率。这项研究表明，设计师的糖肽技术用于商业生产有价值的蛋白质与植物细胞培养物的巨大潜力。
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.