Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.

Exploring the Mutations on Improving Oxidative Stability of Tobacco Etch Virus Protease for Tag-removal in Refolding of Two Disulfide-rich Proteins

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tag, but wild type TEVp shows less oxidative stability, which limits its application under the oxidized redox state to facilitate disulfide bonds formation for refolding disulfide-bonded proteins. Previously, we combined six mutations into the TEVp to generate the TEVp5M for obviously increasing the protein solubility and decreasing the auto-cleavage. In this work, we introduced and combined C19S, C110S and C130S mutations into the TEVp5M to generate seven variants, analyzed protein solubility and the cleavage activity of the constructs in each of three E. coli strains including BL21(DE3), BL21(DE3)pLys, and Rossetta(DE3), and those of the optimized soluble variants in the oxidative cytoplasm of Origami(DE3) under the same induction conditions. The results suggested that desirable protein solubility, cleavage activity and oxidative stability are not combined. Unlike that of the C19S, introduction of the C110S and/or C130S less affected protein solubility but increased tolerance to the oxidative redox state. Use of the TEVp5MC110S/C130S variant, the refolded disulfide-rich bovine enteropeptidase or maize peroxidase was released via cleaving the sequence between the target protein and the cellulose-binding module bound to regenerated amorphous cellulose.

Three Strains of Tobacco etch virus Distinctly Alter the Transcriptome of Apical Stem Tissue in Capsicum annuum during Infection

Tobacco etch virus (TEV; genus Potyvirus) is flexuous rod shaped with a single molecule of single-stranded RNA and causes serious yield losses in species in the Solanaceae. Three TEV strains (HAT, Mex21, and N) are genetically distinct and cause different disease symptoms in plants. Here, a transcriptomic RNA sequencing approach was taken for each TEV strain to evaluate gene expression of the apical stem segment of pepper plants during two stages of disease development. Distinct profiles of Differentially Expressed Genes (DEGs) were identified for each TEV strain. DEG numbers increased with degree of symptom severity: 24 from HAT, 1190 from Mex21, and 4010 from N. At 7 days post-inoculation (dpi), when systemic symptoms were similar, there were few DEGs for HAT- and Mex21-infected plants, whereas N-infected plants had 2516 DEGs. DEG patterns from 7 to 14 dpi corresponded to severity of disease symptoms: milder disease with smaller DEG changes for HAT and Mex21 and severe disease with larger DEG changes for N. Strikingly, in each of these comparisons, there are very few overlapping DEGs among the TEV strains, including no overlapping DEGs between all three strains at 7 or 14 dpi.

Selection of tobacco etch virus protease variants with enhanced oxidative stability for tag-removal in refolding of two disulfide-rich proteins

Tobacco etch virus protease (TEVp) is a powerful enzymatic reagent for removing fusion tag. In this work, we constructed nine TEVp variants with introducing one to three mutations of C19S, C110S and C130S into the soluble TEVp variant, TEVp5M. Using the C-terminal green fluorescent protein (GFP) variant reporter, all constructs showed different solubility levels among four E. coli strains. The TEVp5M containing the C110S and/or C130S mutations in the hyperoxic strain showed the enhanced the cleavage activity. Addition of dithiothreitol to the cultural medium increased the activity of certain constructs produced in the BL21(DE3), contrary to the added hydrogen peroxide, due to cytoplasmic redox change measured by the redox sensitive GFP construct. The more cysteine residues in the purified TEVp5M were modified specifically than those in the other variants. All purified constructs showed similar specific activities in the presence of 5 mM dithiothreitol. In the buffer containing the compounds to aid disulfide bond formation of the refolded protein, the double mutant TEVp5MC110S/C130S exhibited the highest cleavage efficiency. This variant was efficient for removing the fusion tag after refolding of cellulose-binding module tagged disulfide-rich proteins including bovine enteropeptidase and maize peroxidase absorbed on the regenerated amorphous cellulose.

Bacterial expression and protein purification of mini-fluorescence-activating proteins

Bacterial expression and purification of de novo designed mini-fluorescence-activating proteins (mFAPs) are accomplished by genetically fusing mFAP variants to an N-terminal 6xHis tag and optionally to a Tobacco Etch Virus (TEV) protease epitope. The protocol can take less than 24 hours to complete, and allows for downstream in vitro characterization of photophysical properties of mFAPs by equilibration with exogenous fluorogenic compounds.

EFFECT OF Tobacco etch virus (TEV) ON YIELD AND QUALITY OF RED PEPPER IN TURKEY

The most prevalently grown varieties of red peppers in the Eastern Mediterranean region of Turkey are ‘Sena’ and ‘Dila’ in addition to local red pepper populations. Survey studies conducted on Kahramanmaras pepper growing areas in 2014 and 2015 indicated that Tobacco etch virus (TEV) was the most common virus in collected pepper samples. In this study, the effects of TEV on ‘Sena’ and ‘Dila’ were analyzed. The experiment was designed with 5 replicates and randomized plots in fully controlled greenhouses. The experiment consisted of TEV inoculated and control pepper plots. The pepper plants were mechanically inoculated with TEV at the 4–6 leaf stage and periodical observations were made. Virus transmission was confirmed using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) method. The total yield, red pepper flake production, average fruit weight, diameter, length and volume, average fruit wall thickness, fruit color and fresh and dry weights of all green parts of harvested red peppers were evaluated. The quoted data on % reduction in yield and different fruit quality criteria are averaged over two years. According to the results of the study, the highest loss of yield was recorded for ‘Sena’ (58.2%) while the highest red pepper flake loss ratio was in ‘Dila’. In terms of fruit quality criteria, the most reductions in fruit weight (40.1%), fruit diameter (30.9%), fruit length (32.8%) and fruit volume (51.8%) were found in ‘Dila’, the highest losses in fruit wall thickness (27.2%) and average fresh and dry weights of green parts (49.9–43.1%) were in the ‘Sena’. There was a significant effect of TEV inoculation. Overall, virus infected plants were had significantly lower yield and reduced quality compared to control plants.