Hydrophobin Fusions for High-Level Transient Protein Expression and Purification in Nicotiana benthamiana
Hydrophobin Fusions for High-Level Transient Protein Expression and Purification in Nicotiana benthamiana
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DOI：
10.1104/pp.109.149021
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发表时间：
2010-02-01
期刊：
影响因子：
7.4
通讯作者：
Menassa, Rima
中科院分区：
文献类型：
--
作者：
Joensuu, Jussi J.;Conley, Andrew J.;Menassa, Rima
重组蛋白在植物中的积累水平不足，以及缺乏有效的纯化方法来回收这些有价值的蛋白质，阻碍了植物生物技术应用的发展。疏水蛋白是一类来源于丝状真菌的小分子表面活性蛋白，可以通过表面活性剂的双水相系统进行分离纯化。本研究将里氏木霉疏水蛋白HFBI序列与绿色荧光蛋白（GFP）融合，通过农杆菌介导的方法在烟草中进行瞬时表达。HFBI融合显著增强了GFP的积累，融合蛋白的浓度达到总可溶性蛋白的51%，同时还延迟了浸润叶片的坏死。此外，内质网靶向的GFP-HFBI融合诱导了大的新蛋白体的形成。一个简单的和可扩展的基于表面活性剂的双水相系统进行了优化，以回收HFBI融合蛋白的叶提取物。一步相分离能够选择性地回收高达91%的GFP-HFBI，浓度高达10 mg/mL（-1）。HFBI融合增加了植物产生的重组蛋白的表达水平，同时也为其随后的纯化提供了简单的手段。这种疏水蛋白融合技术，当与植物的速度和翻译后修饰能力相结合时，增强了基于植物的瞬时表达系统的价值。
Insufficient accumulation levels of recombinant proteins in plants and the lack of efficient purification methods for recovering these valuable proteins have hindered the development of plant biotechnology applications. Hydrophobins are small and surface-active proteins derived from filamentous fungi that can be easily purified by a surfactant-based aqueous two-phase system. In this study, the hydrophobin HFBI sequence from Trichoderma reesei was fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants by Agrobacterium tumefaciens infiltration. The HFBI fusion significantly enhanced the accumulation of GFP, with the concentration of the fusion protein reaching 51% of total soluble protein, while also delaying necrosis of the infiltrated leaves. Furthermore, the endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of large novel protein bodies. A simple and scalable surfactant-based aqueous two-phase system was optimized to recover the HFBI fusion proteins from leaf extracts. The single-step phase separation was able to selectively recover up to 91% of the GFP-HFBI up to concentrations of 10 mg mL(-1). HFBI fusions increased the expression levels of plant-made recombinant proteins while also providing a simple means for their subsequent purification. This hydrophobin fusion technology, when combined with the speed and posttranslational modification capabilities of plants, enhances the value of transient plant-based expression systems.