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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.
SpyDisplay: A versatile phage display selection system using SpyTag/SpyCatcher technology
Published in mAbs, 2023
Sarah-Jane Kellmann, Christian Hentrich, Mateusz Putyrski, Hanh Hanuschka, Manuel Cavada, Achim Knappik, Francisco Ylera
Fabs were expressed and purified as described previously.27 In short, 2xYT/0.1%Glc/Cam medium was inoculated from an overnight culture with E. coli SK13 containing the expression plasmids and cultivated at 30°C and 250 rpm until the OD600 reached about 0.5, followed by induction with 0.8 mM IPTG and overnight incubation at 27.5°C. Bacterial pellets were lysed with BugBuster (Merck KGaA), supplemented with 20 units/mL Benzonase (Merck KGaA), 2 mg/mL lysozyme (Merck KGaA) and protease inhibitors (Complete EDTA free; Roche) and loaded on Ni-NTA agarose (Qiagen). After washing, Fab fragments were eluted with imidazole-containing buffer (250 mM imidazole, 500 mM NaCl, 20 mM NaH2PO4 pH 7.4), followed by buffer exchange to PBS via PD10 columns (GE Healthcare). TEV protease was expressed and purified as described.49
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].
Discovery of the SHP2 allosteric inhibitor 2-((3R,4R)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-(2,3-dichlorophenyl)-3-methylpyrrolo[2,1-f][1,2,4] triazin-4(3H)-one
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Yanmei Luo, Jin Li, Yuliang Zong, Mengxin Sun, Wan Zheng, Jiapeng Zhu, Liu Liu, Bing Liu
The gene sequence encoding SHP2 residues Met1-Leu525 (UniProt accession code Q06124) was synthesised by GenScript (GenScript.com) with codon optimisation for E. coli expression and inserted into pET-15b containing an N-Terminal 6 × His-tag and a tobacco etch virus (TEV) cleavage site. The E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown in Luria-Bertani medium at 37 °C until OD600 reached 0.6–0.8. Protein expression was induced by isopropyl-β-D-thiogalactoside to a final concentration of 0.2 mM and the cells were grown overnight at 18 °C and then harvested by centrifugation at 6000g for 15 min at 4 °C. The cell pellet was re-suspended in buffer A (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% glycerol) and lysed by a high-pressure homogeniser at 1000 bar. Cell debris was removed by centrifugation at 47,000g for 30 min. The supernatant was loaded onto a 5 mL Ni-NTA column (GE Healthcare), which had been equilibrated in buffer A. A gradient wash was applied with increasing concentration of imidazole (10–50 mM) in the same buffer in order to remove non-specific bound protein. The His-tagged SHP2 was eluted with 250 mM imidazole in the same buffer. The 6 × His-tag was cleaved by TEV protease overnight at 4 °C. The TEV protease and uncleaved 6 × His-tagged protein were removed by passing through the Ni-NTA column again. The flow-through containing His-tag removed SHP2 was concentrated and further purified by a size-exclusion column Superose 12 10/300 (GE Healthcare) and the purest fraction of SHP2 was concentrated to ∼12 mg/mL in 5 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM DTT.