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首页> 外文期刊>Journal of structural and functional genomics >Functional protein expression from a DNA based wheat germ cell-free system
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Functional protein expression from a DNA based wheat germ cell-free system

机译：来自基于DNA的小麦胚无细胞系统的功能蛋白表达

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Wheat germ based eukaryotic cell-free systems have been shown to be applicable for both functional and structural analyses of proteins. However, the existing methods might require specialized instrumentation and/or a separate mRNA synthesis step. We have developed a DNA based, highly productive, coupled transcription/translation wheat germ cell-free system that incorporates the normally separate mRNA synthesis step and does not require specialized instrumentation. Using a small-volume batch reaction with fluorescence labeling, DNA templates predicted to encode proteins could be quickly screened for their ability to direct the expression of proteins of the appropriate size. Protein yield can be increased as much as 2 to 4-fold in this system using a dialysis reaction, reaching ～200–440?μg/ml in 10–20?h. Furthermore, enzyme activities can be assayed directly in the extract without further purification. Simple purification with affinity tags can be achieved in one-step and with minor modifications, efficient SeMet and [U-15N] labeling of >95% can be accomplished in this system. Thus, this efficient cell-free expression system can facilitate both functional and structural proteomics.
机译：已显示基于小麦胚芽的真核无细胞系统可用于蛋白质的功能和结构分析。但是，现有方法可能需要专门的仪器和/或单独的mRNA合成步骤。我们已经开发出一种基于DNA的，高产的，耦合的转录/翻译小麦无细胞系统，该系统整合了通常独立的mRNA合成步骤，并且不需要专门的仪器。使用带有荧光标记的小批量分批反应，可以快速筛选出预测为编码蛋白质的DNA模板指导适当大小的蛋白质表达的能力。通过透析反应，该系统中的蛋白质产量可以增加2到4倍，在10-20小时内达到〜200-440？μg/ ml。此外，无需进一步纯化即可直接在提取物中测定酶活性。只需一步即可完成具有亲和标签的简单纯化，而只需进行少量修改，即可在该系统中完成高效的SeMet和[U-15N]标记> 95％。因此，这种有效的无细胞表达系统可以促进功能和结构蛋白质组学。

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