Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 37-47 Tűzoltó Street, Budapest, H-1094, Hungary.
BMC Biotechnol. 2020 Mar 14;20(1):17. doi: 10.1186/s12896-020-00610-5.
Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively.
Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies.
The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.
无细胞蛋白质表达已成为功能和结构研究中替代体内基于细胞的系统的广泛应用方法。基于小麦胚的方法通过其灵活性、高翻译效率和正确折叠的真核蛋白质合成的成功率，从商业上可用的真核体外翻译系统中脱颖而出。原始的含有 T7 启动子的 pEU3-NII 载体通过添加无连接克隆位点、His 和 GST 标签以及 TEV 蛋白酶切割位点得到了改进，这分别有利于重组质粒的创建、允许亲和纯化以及产生纯化的、无标签的目标蛋白。
在这里，我们通过插入稀有切割的 NotI 限制酶切割位点进一步开发了 pEU3-NII 载体，以便在体外转录之前简化载体线性化步骤。此外，还创建了 His、FLAG 和 Halo 亲和标签编码载体，以分别提高检测灵敏度、相互作用研究的特异性，并提供用于下拉测定的共价连接配体。最后，所提出的 GST-His 和 GST-生物素双标记载体可以拓宽蛋白质-蛋白质相互作用研究的可能性范围。
新一代 pEU3-NII 载体家族允许更快速地生产翻译活性的 mRNA 和小麦胚无细胞表达目标蛋白，具有多种亲和标签，从而能够为体外蛋白质研究设计灵活多样的实验方案。
