Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition
Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition
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带有设计糖肽标签的烟草细胞中表达的重组蛋白的显着分泌受到培养基成分的高度影响
DOI：
10.1007/s00299-016-2051-6
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发表时间：
2016
影响因子：
6.2
通讯作者：
Xu, Jianfeng
中科院分区：
文献类型：
--
作者：
Zhang, Ningning;Dolan, Maureen;Wu, Di;Phillips, Gregory C.;Xu, Jianfeng
细胞生长培养基组成对“设计”阿拉伯半乳聚糖蛋白基模块的糖基化有深远的影响；充分的糖基化是指导有效的细胞外分泌标记重组蛋白的必要条件。摘要将重组蛋白与新设计的羟脯氨酸(Hyp)- o糖基化肽（HypGP）标签融合在植物细胞中表达，即HypGP工程技术，可显著提高分泌蛋白的产量。这是由于HypGP标签作为分子载体的功能，促进连接蛋白有效地运输到培养基中。为了优化细胞培养以获得最佳的分泌蛋白产量，以烟草BY-2细胞为模型系统，研究了培养基对细胞生长和蛋白分泌的影响，该细胞表达增强绿色荧光蛋白（EGFP）与（SP）32标签（Ser-Pro motif的32个串联重复序列）融合。发现（SP）32tag在植物细胞中经历了两阶段的hypo糖基化，与之相连的EGFP的大量分泌与第二阶段糖基化的触发相关。在SH培养基中培养的BY-2细胞分泌蛋白产量高（125 mg/L），细胞生物量积累低（~7.5 gDW/L）。相比之下，在MS培养基中获得了极低的分泌蛋白产量（~1.5 mg/L）和高细胞生物量积累（13.5 gDW/L）。宏量营养素，特别是氮的供应，极大地影响了（SP）32标签的糖基化和随后的蛋白质分泌。减少氮含量的改良MS培养基使EGFP产量提高到168 mg/L。本研究证明了培养基组成对HypGP标记蛋白分泌量的深远影响，为培养基设计提供了基础，以实现HypGP工程技术的最高产量。
Key messageCell growth medium composition has profound impacts on theO-glycosylation of a “designer” arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein.AbstractExpression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)32tag (32 tandem repeats of “Ser-Pro” motif). The (SP)32tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with thetriggeringof the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)32tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.
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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。
影响因子：
4.7
作者：
Zhang,Ningning;Gonzalez,Maria;Savary,Brett;Xu,Jianfeng
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Xu,Jianfeng
影响因子：
13.8
作者：
Wilson SA;Roberts SC
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影响因子：
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作者：
Bonny M.‐Y. Tsoi;Pauline M. Doran
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影响因子：
4.3
作者：
GRIMES, HD;HODGES, TK
通讯作者：
HODGES, TK
影响因子：
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作者：
Dolan, Maureen C.;Wu, Di;Xu, Jianfeng
通讯作者：
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