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Order Patatavirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Although not directly connected with the VLP story, it should be noted that the first potyvirus-based vector system for the foreign gene expression in planta was elaborated on tobacco etch virus (TEV) by Dolja et al. (1992), after the efficient vector systems elaborated earlier in the 1980s and based on brome mosaic virus (BMV), order Martellivirales, family Bromoviridae (Chapter 17); tobacco mosaic virus (TMV), order Martellivirales, family Virgaviridae (Chapter 19); and potato virus X (PVX), order Tymovirales (Chapter 21). After TEV, other potyviruses followed as subjects by the elaboration of the in planta expression vectors, namely plum pox virus (PPV; Guo et al. 1998), lettuce mosaic virus (LMV; German-Retana et al. 2000), clover yellow vein virus (ClYVV; Masuta et al. 2000), pea seed-borne mosaic virus (PSbMV; Johansen et al. 2001), zucchini yellow mosaic virus (ZYMV; Arazi et al. 2001b, 2002; Hsu et al. 2004), turnip mosaic potyvirus (TuMV; Beauchemin et al. 2005; Chen et al. 2007; Touriño et al. 2008), and potato virus A (Kelloniemi et al. 2008). From the general point of development of the advanced nanotechnological applications, the well-known and universally used TEV protease (Kapust and Waugh 2000) is also worth mentioning here.
Expression
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Concerning the crystal structures of the Qβ replicase complex, which were described in detail in the Fine structure of the Qβ replicase section of Chapter 13, Kidmose et al. (2010) operated with the enzyme purified from the E. coli strain carrying the single Ts-Tu-β expression vector, according to Kita et al. (2006). The latter was modified to insert a tobacco etch virus (TEV) protease cleavage site in the fusion protein EFTs–EFTu–TEV–βS–6xHis. The E. coli BL21 (DE3) strain was transformed with the resulting plasmid, grown in LB medium, and induced with L-arabinose. The fusion protein was purified by Ni2+ chelate chromatography and subsequently digested by TEV protease. The cleaved fusion protein was further purified by hydrophobic interaction chromatography and gel filtration. The complex of the subunit β, or II, and EF-TuTs was resolved then at 2.5 Å resolution (Kidmose et al. 2010).
EspH interacts with the host active Bcr related (ABR) protein to suppress RhoGTPases
Published in Gut Microbes, 2022
Rachana Pattani Ramachandran, Ipsita Nandi, Nir Haritan, Efrat Zlotkin-Rivkin, Yael Keren, Tsafi Danieli, Mario Lebendiker, Naomi Melamed-Book, William Breuer, Dana Reichmann, Benjamin Aroeti
Finally, we asked whether the EspH-38aa segment can interact with ABR. Initially, pulldown assays using purified glutathione s transferase (GST)-EspH-38aa-6xHis-streptavidin (StAv) binding peptide (SBP) coupled to StAv beads failed to coprecipitate the endogenous ABR from HeLa cells (data not shown). This has raised the possibility that the N-terminally fused GST interfered with ABR binding. Therefore, similar pulldown experiments were performed after GST removal by the tobacco etch virus (TEV) protease cleavage, i.e., using purified EspH-38aa-6xHis-SBP (Figure S2) as a bait. Indeed, unlike the StAv beads alone, EspH-38aa-6xHis-SBP coupled to StAv beads precipitated the ABR from cell lysates (Figure 1e). Similarly, the EspH-38aa-6xHis-SBP fragment could coprecipitate the purified human 6xHis-TEV-SUMO-ABR-2xFLAG (Figure S3), but not the purified 6xHis-TEV-SUMO-eGFP (Figure S4) control (figure 1f). Taken together, these results suggest that translocated EspH interacts firmly with ABR and that the C-terminal 38-aa segment of the effector plays a role in mediating these interactions.
Zika virus pathogenesis and current therapeutic advances
Published in Pathogens and Global Health, 2021
Caroline Mwaliko, Raphael Nyaruaba, Lu Zhao, Evans Atoni, Samuel Karungu, Matilu Mwau, Dimitri Lavillette, Han Xia, Zhiming Yuan
Kim et al. designed a recombinant E1/E3-deleted adenoviral vector (pAd.ZIKV-Efl) from the ZIKV strain BeH815744 that expressed a codon-optimized Env-antigen. The extracellular region of the ZIKV envelope was linked to the T4 fibrin trimerization domain, facilitating protein expression. Additionally, the vector was designed with a polyhistidine tag and a Tobacco Etch Virus (TEV) to increase protein folding and facilitate purification. The vaccine was shown to protect C57BL/6 mice from lethal challenge with the ZIKV DAKAR41542 strain [100]. Notably, in these vaccine studies, the ZIKV E subunit protein production yield was very low, a finding that has also been highlighted by Larroca et al. [93]. The low yield of the E protein was probably due to the absence of preM, which is important for protein stability. Therefore, preM is an indispensable factor in the development of ZIKV E protein-based vaccines.
Protein amino-termini and how to identify them
Published in Expert Review of Proteomics, 2020
Annelies Bogaert, Kris Gevaert
Essential when directly enriching for N-terminal peptides are labeling methods that are highly selective for α-amines over ε- amines of lysine side-chains. One such labeling method was developed by the Wells lab, who engineered a ligase, termed subtiligase, a variant of the subtilisin protease, which exhibits absolute selectivity for primary (non-blocked) α-amines [69]. Proteomes are treated with subtiligase and a peptide glycolate ester substrate specifically tailored for N-terminomics, leading to the enzymatic biotinylation of protein N-termini. Subsequently, proteomes are digested and the biontinylated N-terminal peptides are captured by avidin beads. A tobacco etch virus (TEV) protease cleavage site present in the biotin-ligated peptides allows selective recovery of N-terminal peptides from the avidin beads (Figure 4). In the original protocol, these recovered N-terminal peptides contained an SY-dipeptide remnant after TEV protease cleavage, which readily allowed to differentiate between captured N-terminal peptides and co-eluted, contaminating peptides following LC-MS/MS data analysis [21]. In later versions of the method, the SY-dipeptide motif was changed for an aminobutyric acid residue, which led to less interference of the SY-tag fragment ions in MS/MS spectra and an MS-discernable, non-natural mass signature [70].