plasmid encoding t7 promoter  (Addgene inc)

Journal: bioRxiv

Article Title: Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats

doi: 10.1101/2023.03.02.530738

Figure Lengend Snippet: ( A ) V976L mutation emerged in 190366 virus stocks. ( B ) Pseudotyped particles were produced with full-length spike WT or the V967L mutant. Spike was detected in producer cells and pseudotyped by western blotting for FLAG. ( C ) Indicated cells were infected with viral pseudotyped in the presence or absence of trypsin. ( D ) Schematic overview of the dual-reporter fusion assay developed for this study. T7 polymerase drives expression of GFP and luciferase separated by a P2A fusion peptide. ( E ) HEK 293T cells expressing receptor or, ( F) empty vector and T7-polymerase were combined with cells expressing spike and the T7-driven reporter. Luciferase was measured as a readout for cell fusion. Dotted lines indicate data from 1:4 ratio of receptor:spike cells. ( G ) Overview of 190366 spike with in silico predicted trypsin digest sites indicated. Location of V976L is indicated in green. ( H ) Concentrated viral pseudotyped were combined with a wide range or trypsin dilutions or ( I ) a fine range of trypsin dilutions and incubated at 37°C. Spike digestion was assessed by western blot for FLAG epitope.

Article Snippet: Plasmid encoding T7-promoter driven dual reporter GFP and luciferase was generated by cloning firefly luciferase downstream of GFP in pUC19-T7-IRES-GFP. pUC19 - T7 pro - IRES - EGFP was a gift from Fei Chen (Addgene plasmid # 138586; http://n2t.net/addgene:138586 ; RRID: Addgene_138586).

Techniques: Mutagenesis, Produced, Western Blot, Infection, Single Vesicle Fusion Assay, Expressing, Luciferase, Plasmid Preparation, In Silico, Incubation, FLAG-tag

Journal:

Article Title: Deletion of Ser-171 causes inactivation, proteasome-mediated degradation and complete deficiency of human transaldolase

doi: 10.1042/BJ20040413

Figure Lengend Snippet: (A) Prokaryotic expressions of wild-type and mutant TALΔ561–563 cDNAs. The TAL-H protein was expressed as a fusion protein with GST encoded by pGEX-2T plasmid vector [11]. Maximal expression of the recombinant fusion protein was obtained after stimulation with 1 mM IPTG for 2 h. TAL-H–GST fusion protein was affinity-purified through binding of GST to glutathione-coated agarose beads. Protein lysates were analysed on 12% SDS/polyacrylamide gel. Lane 1, whole cell lysates from unstimulated cells; lane 2, whole cell lysates from IPTG-stimulated cells; lane 3, supernatant of IPTG-stimulated cells disrupted by freezing and thawing performed three times; lane 4, pellet of IPTG-stimulated cells disrupted by freezing and thawing performed three times; lane 5, supernatant of IPTG-stimulated cells incubated with GSH-coated agarose beads; lane 6, GSH-coated agarose beads pelleted after exposure to IPTG-stimulated cell supernatant. (B) Western-blot detection of solubilized, affinity-purified and thrombin-cleaved recombinant TAL–GST fusion proteins. IPTG-stimulated cells were disrupted in the absence or presence of 1.5% N-laurylsarcosine, 2% Tween 20 and 4% Triton X-100, and supernatants were incubated with GSH-coated agarose beads. Subsequently, beads were washed six times in 1 ml of PBS, digested overnight with thrombin in 500 μl of PBS and tested for the presence of TAL by Western blotting using antibody 170. Lane 1, whole cell lysate from unstimulated cells; lane 2, whole cell lysate from IPTG-stimulated cells; lane 3, GSH-coated agarose beads exposed to the supernatant of IPTG-stimulated cells disrupted by freezing and thawing performed three times; lane 4, supernatant of thrombin-treated agarose beads previously exposed to the supernatant of IPTG-stimulated cells disrupted by freezing and thawing three times; 5, GSH-coated agarose beads exposed to the supernatant of IPTG-stimulated cells disrupted by freezing and thawing three times in the presence of 1.5% N-laurylsarcosine, 2% Tween 20 and 4% Triton X-100; lane 6, supernatant of thrombin-treated agarose beads previously exposed to the supernatant of IPTG-stimulated cells disrupted by freezing and thawing three times in the presence of 1.5% N-laurylsarcosine, 2% Tween 20 and 4% Triton X-100. (C) In vitro translation of wild-type and mutant TALΔ561–563 RNA by rabbit reticulocyte lysates. For generating the [35S]methionine-labelled product, 1 μg of pCMVTNT-based wild-type or mutant TAL-H cDNA was mixed with nuclease-free rabbit reticulocyte lysate, T7 RNA polymerase, amino acids minus methionine and 20 μCi of L-[35S]methionine and incubated at 30 °C for 90 min. As indicated, firefly luciferase-encoding control plasmid driven by the T7 RNA polymerase promoter was added to the reaction mixture.

Article Snippet: The reaction was carried out at 30 °C for 90 min. Where indicated, 1 μg of firefly luciferase-encoding control plasmid driven by the T7 RNA polymerase promoter (Promega) was added to the reaction mixture.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Recombinant, Affinity Purification, Binding Assay, Incubation, Western Blot, In Vitro, Luciferase