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

Abstract

Background: 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 6-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. Results: 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 12 , 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 6 , and GSTbiotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. Conclusions: 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.

Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

“…The Drosophila nucleoplasmin, dNLP, coding gene was amplified from the cDNA library of adult Drosophila whole body, then cloned into the pEU-E01-MCS vector. The human core histones were amplified from the human cDNA library and cloned into a pEU-E01-LICNot expression vector [ 41 ]. The pEU vector used in this study has been optimized to maximize the efficiency of in vitro wheat germ-based cell-free protein synthesis [ 41 , 42 ].…”

“…The human core histones were amplified from the human cDNA library and cloned into a pEU-E01-LICNot expression vector [ 41 ]. The pEU vector used in this study has been optimized to maximize the efficiency of in vitro wheat germ-based cell-free protein synthesis [ 41 , 42 ]. The applied vectors do not possess an affinity tag coding region since we aimed to achieve the physiological state of histones and a single-step nucleosome assembly without affinity purification.…”

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

“…In contrast, the Strep-tag II (Schmidt and Skerra, 2007) allows for lower cost affinity purification, with high purity levels reached already after a single purification step. It uses a (Malhotra, 2009;Vinarov et al, 2006a;Aoki et al, 2009;Revathi et al, 2010;Takahashi et al, 2012) (Aceti et al, 2015;Li et al, 2016;Novikova et al, 2018) (Fogeron et al, 2015a;Fogeron et al, 2015b;Li et al, 2016;Fogeron et al, 2017a;Minkoff et al, 2017) Cost (Einhauer and Jungbauer, 2001;Bardoczy et al, 2008;Ramadan et al, 2015;Novikova et al, 2018;Nagy et al, 2020) No binding of WGE endogenous proteins to the affinity support elution by enzymatic cleavage or by competition high protein recovery and high purity level in a only one-step purification process Lower binding capacity of the affinity support Higher purification cost than for other tags C-ter (Novikova et al, 2018) Streptag II Purification by affinity chromatography N-ter (Schmidt and Scerra, 2007;David et al, 2019) No binding of WGE endogenous proteins to the affinity support Cost-effective and easy purification process Elution by competition under native conditions Affinity support reusable up to five times Slightly lower binding capacity of the affinity support than for His-and GST-tags Higher purification cost than for His-and GST-tags C-ter (Fogeron et al, 2015a;Fogeron et al, 2015b;Li et al, 2016;Fogeron et al, 2017a;Minkoff et al, 2017; minimal peptide sequence (WSHPQFEK) with a high affinity to native streptavidin or an engineered streptavidin having even higher affinity for the tag (Strep-Tactin (Maertens et al, 2015)).…”

“…When even higher affinity is needed during purification, a Twin-Strep-tag (Schmidt et al, 2013) is available that can also be used with WGS (Fogeron et al, 2016;Boukadida et al, 2018;Jirasko et al, 2020). The use of the HaloTag, a 297 amino acid peptide derived from a bacterial haloalkane dehalogenase (Los et al, 2008), has been recently described for the WGS (Nagy et al, 2020). Because the HaloTag forms a highly specific covalent bond with its synthetic ligand, this tag is of particular interest for pull-down assays, allowing for more stringent buffer conditions and washing steps (Los et al, 2008).…”

“…Most fusion proteins have added sequences encoding an affinity tag that can be added at either end of the cDNA; small tags may also be added by primer extension PCR. Ready-to-use commercial expression vectors are available for the WGS or have been described in the literature ( Bardoczy et al, 2008 ; Nagy et al, 2020 ). While affinity tags can be especially useful in protein purification, they also offer means for protein detection and analysis ( Kimple et al, 2013 ; Wood, 2014 ; Yadav et al, 2016 ).…”