Division of Molecular and Life Sciences and Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, 37673, Korea.
Sci Rep. 2018 Mar 15;8(1):4612. doi: 10.1038/s41598-018-22860-2.
Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on protein production and provide evidence that N-glycosylation greatly increases the expression levels of ER-targeted recombinant proteins. Fusion of the extracellular domain (M domain) of protein tyrosine phosphatase receptor type C (CD45), which contains four putative N-glycosylation sites to a model protein, leptin at the C-terminus, increased recombinant protein levels by 6.1 fold. This increase was specific to ER-targeted proteins and was dependent on N-glycosylation. Moreover, expression levels of leptin, leukemia inhibitory factor and GFP were also greatly increased by fusion of M domain at either the N or C-terminus. Furthermore, the increase in protein levels resulted from enhanced translation, but not transcription. Based on these results, we propose that fusing a small domain containing N-glycosylation sites to target proteins is a powerful technique for increasing the expression levels of recombinant proteins in plants.
植物是生产各种重组蛋白的有前途的系统。改进这项技术的一个重点领域是开发高水平生产重组蛋白的方法。已经开发出许多方法来提高重组基因的转录水平。然而，涉及转录后步骤（包括翻译）的增加蛋白质产量的方法尚未得到充分探索。在这里，我们研究了 N-糖基化对蛋白质生产的影响，并提供了证据表明 N-糖基化大大增加了内质网靶向重组蛋白的表达水平。将蛋白酪氨酸磷酸酶受体 C（CD45）的细胞外结构域（M 结构域）与模型蛋白瘦素在 C 末端融合，该结构域包含四个假定的 N-糖基化位点，将重组蛋白水平提高了 6.1 倍。这种增加是内质网靶向蛋白特有的，并且依赖于 N-糖基化。此外，通过在 N 或 C 末端融合 M 结构域，瘦素、白血病抑制因子和 GFP 的表达水平也大大增加。此外，蛋白质水平的增加是由于翻译增强，而不是转录增强。基于这些结果，我们提出将含有 N-糖基化位点的小结构域融合到靶蛋白上是一种在植物中提高重组蛋白表达水平的有效技术。
