Immunogenic properties of empty pcDNA3 plasmid against zoonotic cutaneous leishmaniasis in mice

Montakhab-Yeganeh, Hossein; Salehi, Reza; Najm, Mehdi; Masoori, Leila; Aspatwar, Ashok; Badirzadeh, Alireza

doi:10.1371/journal.pone.0263993

Cited by 2 publications

(1 citation statement)

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“…All of the split TEV assays were based on transient transfection with pcDNA3-based vectors, which are some of the most frequently used vectors in molecular and cellular biology [35]. However, we cannot rule out any differences in assay performance that may occur when using different vector backbones or different promoters (we used the CMV promoter; other options are, e.g., human EF1α or UbC promoters) for expressing split TEV fusion proteins.…”

Section: Discussionmentioning

confidence: 99%

Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants

Hauff

Jensen

et al. 2022

Biosensors

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G protein-coupled receptors (GPCRs) are major disease-relevant drug targets; robust monitoring of their activities upon drug treatment is key to drug discovery. The split TEV cell-based assay technique monitors the interaction of an activated GPCR with β-arrestin-2 through TEV protein fragment complementation using a luminescent signal as the readout. In this work, split TEV GPCR β-arrestin-2 recruitment assays were optimized to monitor the endogenous ligand-induced activities of six GPCRs (DRD1, DRD2, HTR2A, GCGR, AVPR2, and GLP1R). Each GPCR was tested in four forms; i.e., its wildtype form, a variant with a signal peptide (SP) to facilitate receptor expression, a variant containing the C-terminal tail from the V2 vasopressin receptor (V2R tail) to promote β-arrestin-2 recruitment, and a variant containing both the SP and V2R tail. These 24 GPCR variants were systematically tested for assay performance in four cell lines (HEK-293, PC12 Tet-Off, U-2 OS, and HeLa). We found that the assay performance differed significantly for each GPCR variant and was dependent on the cell line. We found that V2R improved the DRD2 split TEV assays and that HEK-293 cells were the preferred cell line across the GPCRs tested. When taking these considerations into account, the defined selection of assay modifications and conditions may improve the performance of drug development campaigns that apply the split TEV technique as a screening tool.

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Section: Discussionmentioning

confidence: 99%