Production of Bioactive Recombinant Reteplase by Virus-Based Transient Expression System in Nicotiana benthamiana
Production of Bioactive Recombinant Reteplase by Virus-Based Transient Expression System in Nicotiana benthamiana
复制标题
利用本塞姆氏烟草病毒瞬时表达系统生产生物活性重组 rPA（瑞替普酶）
DOI：
10.3389/fpls.2019.01225
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发表时间：
2019-10-08
影响因子：
5.6
通讯作者：
Wang, Sheng
中科院分区：
文献类型：
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作者：
Ma, Ting;Li, Zhiying;Wang, Sheng
为了探索一种经济高效的替代方法生产重组溶栓药物Reteplase (rPA)，采用基于植物病毒扩增子的基因表达系统，通过农业渗透在烟叶中瞬时表达具有生物活性的Strep ii标记的重组rPA。设计并合成了多个基因表达载体，并将其克隆到烟草花叶病毒rna过表达载体上。通过对密码子优化、亚细胞靶向和附着的Strep-tag II的效果进行评估，确定重组rPA在烟叶中表达水平最高的条件。我们发现，以内质网为靶点的带有n端Strep-tag II的密码子优化rPA在不到1周的时间内可从每公斤鲜重叶片生物量中获得高达50 mg的生物活性蛋白。此外，通过Strep-tag II一步纯化，可以方便地从接种的叶片提取物中纯化重组rPA。该植物合成的rPA被糖基化，分子量约为45.0 kDa，其体外纤溶活性与市售rPA相当。这些结果表明，基于植物病毒扩增子的系统为大规模生产具有成本效益的重组rPA提供了一种简单高效的方法。
To explore a cost-effective alternative method to produce the recombinant thrombolytic drug Reteplase (rPA), a plant viral amplicon-based gene expression system was employed to transiently express bioactive Strep II-tagged recombinant rPA in Nicotiana benthamiana leaves via agro-infiltration. Several gene expression cassettes were designed, synthesized in vitro, and then cloned into Tobacco mosaic virus RNA-based overexpression vector. Codon optimization, subcellular targeting, and the effect of attached Strep-tag II were assessed to identify conditions that maximized expression levels of the recombinant rPA in tobacco leaves. We found that codon-optimized rPA with N-terminal Strep-tag II that was aimed to the endoplasmic reticulum as target provided the highest amount of biologically active protein, i.e., up to similar to 50 mg from per kilogram fresh weight leaf biomass in less than 1 week. Furthermore, the recombinant rPA was conveniently purified from inoculated leaf extracts by a one-step purification procedure via the Strep-tag II. The plant-made rPA was glycosylated with molecular mass of similar to 45.0 kDa, and its in vitro fibrinolysis activity was equivalent to the commercial available rPA. These results indicate that the plant viral amplicon-based system offers a simple and highly effective approach for cost-effective large-scale production of recombinant rPA.