A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression.

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, His6- 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, His12, 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-His6, 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.