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Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.

机译:使用基于大肠杆菌的表达系统生成重同位素和硒甲硫氨酸标记的蛋白进行结构测定的优化程序。

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摘要

Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins ((15)N, (13)C, and (2)H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for (15)N and (13)C, respectively, and 75% of (non-exchangeable) hydrogen for (2)H labeling. The expression yields and final cell densities (OD600 ~16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.
机译:产生足够量的标记蛋白质代表蛋白质结构确定中的瓶颈。使用基于T7的大肠杆菌表达系统,开发了一种用于生产重同位素以及硒代蛋氨酸(Se-Met)标记的蛋白的简单方案。该协议适用于在摇瓶培养物中生成单,双和三标记蛋白((15)N,(13)C和(2)H)。对于(15)N和(13)C,标记掺入目标蛋白质的比例分别达到99%和97%,对于(2)H标记,掺入75%的氢(不可交换)。表达产量和最终细胞密度(OD600〜16)与非标记蛋白的产生相同。该方案也适用于Se-Met标记,分别使用原养型或蛋氨酸营养型大肠杆菌菌株将Se-Met掺入目标蛋白的70%或90%。

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