pet3a expression vector  (Thermo Fisher)

Journal: Scientific Reports

Article Title: Multilevel structure–activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

doi: 10.1038/s41598-019-48888-6

Figure Lengend Snippet: Catechin gallates, alkyl gallates, and myricetin inhibit PCNA ubiquitination. ( a ) Representative Western blot probed with anti-FLAG antibody showing the dose–response for inhibition of PCNA ubiquitination by EGCG (0.0122 to 6.25 μM in concentration increments of 2×), with a final DMSO carrier solvent concentration of 2% in all cases. Samples were preincubated with EGCG for 15 min prior to addition of ATP, then further incubated for 2 h. Negative and positive controls consisted of parallel samples incubated with DMSO alone in the absence (−) or presence (+) of ATP, respectively. ( b ) Dose–response for % PCNA ubiquitination for the different compounds, plotted both linearly and semi-logarithmically (inset), with a DMSO concentration of 2% in all cases. Data represent mean ± SD, derived from quantitation of Western blot images according to (FLAG-PCNA–ubiquitin)/(FLAG-PCNA + FLAG-PCNA–ubiquitin) × 100% for each lane internally from 3–7 independent experiments, as indicated for each case in Table along with IC 50 values. ( c ) % PCNA ubiquitination for compounds with no inhibitory activity even at 500 μM, with a DMSO concentration of 2% in all cases. Data represent mean and SD for three independent experiments. Negative and positive controls consisted of DMSO alone without or with ATP, respectively. ( d ) Effects of EGCG and Myr on Uba1 and ubiquitination are reversible. 30 μM EGCG or 30 μM Myr (with DMSO alone as control and a DMSO concentration of 2% in all cases) were preincubated with Uba1 for 2 h, completely washed out by serial centrifugal filtration, and then assayed for PCNA ubiquitination activity after addition of other reaction components, with detection by Western blot analysis with anti-FLAG antibody.

Article Snippet: His-tagged human Uba1 in the pET3a bacterial expression vector (Addgene Cat#63571) was expressed in the E . coli strain BL21(DE3) and purified with Ni-NTA agarose beads (Macherey-Nagel).

Techniques: Western Blot, Inhibition, Concentration Assay, Incubation, Derivative Assay, Quantitation Assay, Activity Assay, Filtration

Journal: Scientific Reports

Article Title: Multilevel structure–activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

doi: 10.1038/s41598-019-48888-6

Figure Lengend Snippet: Half-maximal inhibitory concentration (IC 50 ) values for compounds against PCNA ubiquitination and Uba1~ubiquitin thioester formation and effects on viability of normal, Uba1-overexpressing, and ubiquitin-overexpressing cells.

Article Snippet: His-tagged human Uba1 in the pET3a bacterial expression vector (Addgene Cat#63571) was expressed in the E . coli strain BL21(DE3) and purified with Ni-NTA agarose beads (Macherey-Nagel).

Techniques: Concentration Assay, Inhibition, Over Expression

Journal: Scientific Reports

Article Title: Multilevel structure–activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

doi: 10.1038/s41598-019-48888-6

Figure Lengend Snippet: Structure–activity relationships for inhibition of PCNA ubiquitination, Uba1~ubiquitin thioester formation, and viability of normal, Uba1-overexpressing, and ubiquitin-overexpressing cells. More detailed information is contained in Figs , and and Table . OE = overexpression; N/A = not applicable.

Article Snippet: His-tagged human Uba1 in the pET3a bacterial expression vector (Addgene Cat#63571) was expressed in the E . coli strain BL21(DE3) and purified with Ni-NTA agarose beads (Macherey-Nagel).

Techniques: Activity Assay, Inhibition, Over Expression

Journal: Scientific Reports

Article Title: Multilevel structure–activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

doi: 10.1038/s41598-019-48888-6

Figure Lengend Snippet: Catechin gallates, alkyl gallates, and myricetin inhibit Uba1~ubiquitin thioester formation. ( a ) Representative Western blots showing that EGCG inhibits Uba1~ubiquitin thioester formation when preincubated with Uba1 before adding ubiquitin, but not when preincubated with ubiquitin first, while Myr inhibits Uba1~ubiquitin thioester in both cases. Final compound concentrations were 10 μM for EGCG or 50 μM for Myr (with DMSO alone as control and a DMSO concentration of 2% in all cases). Following preincubation, reactions were allowed to proceed for 2 h with ubiquitin, Uba1, and ATP. Comparison of results in the absence to presence of DTT as reducing agent confirms that the conjugation is through the reducible thioester. ( b ) Dose–response for relative % Uba1~ubiquitin thioester formation with increasing concentrations of each compound, plotted both linearly and semi-logarithmically (inset), with a DMSO concentration of 2% in all cases. Data represent mean ± SD for 3–4 independent experiments, as indicated along with IC 50 values for each case in Table . Each value was normalized on a gel-by-gel basis to the parallel positive control (DMSO alone with ATP) level of Uba1~ubiquitin thioester conjugate formed in each case and from quantitation of silver-stained SDS-polyacrylamide gels of reaction samples without DTT. ( c ) Relative % Uba1~ubiquitin thioester formation for compounds with no inhibitory activity even at 500 μM, with a DMSO concentration of 2% in all cases. Data represent mean and SD for four independent experiments. ( d ) EGCG does not inhibit formation of the Rad6~ubiquitin thioester conjugate resulting from transfer of ubiquitin from already formed Uba1~ubiquitin. The assay, with a DMSO concentration of 2% in all cases, and quantitation were done analogously to those for the Uba1~ubiquitin thioester formation experiments and are described in Methods. Data represent mean and SD for four independent experiments.

Article Snippet: His-tagged human Uba1 in the pET3a bacterial expression vector (Addgene Cat#63571) was expressed in the E . coli strain BL21(DE3) and purified with Ni-NTA agarose beads (Macherey-Nagel).

Techniques: Western Blot, Concentration Assay, Conjugation Assay, Positive Control, Quantitation Assay, Staining, Activity Assay

Journal: Scientific Reports

Article Title: Multilevel structure–activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

doi: 10.1038/s41598-019-48888-6

Figure Lengend Snippet: NMR reveals that EGCG directly binds Uba1. ( a ) Aromatic 1 H NMR region of EGCG in the presence of Uba1 (red) and without protein (black). ( b ) STD NMR spectrum recorded for EGCG in the presence of Uba1, aromatic region. ( c ) TrNOE experiment: no crosspeaks are present in the EGCG control experiment (left), while several crosspeaks appeared in the presence of Uba1 (negative NOE due to an equilibrium between the free and the bound state of EGCG). The data suggest a K d in the range of high nM to low μM, as discussed in Results.

Article Snippet: His-tagged human Uba1 in the pET3a bacterial expression vector (Addgene Cat#63571) was expressed in the E . coli strain BL21(DE3) and purified with Ni-NTA agarose beads (Macherey-Nagel).

Techniques:

Journal: Scientific Reports

Article Title: Multilevel structure–activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

doi: 10.1038/s41598-019-48888-6

Figure Lengend Snippet: Catechin gallates, alkyl gallates, and myricetin reduce cell viability in a manner protected by overexpression of Uba1 or ubiquitin in cells and EGCG inhibits global cellular ubiquitination. ( a ) Dose–response for effects on HEK 293 cell survival after 24-h treatment with each compound relative to parallel untreated controls by the Alamar Blue (resazurin) assay. Data represent mean ± SD for 4–6 independent experiments (as indicated along with IC 50 values for each case in Table ), plotted both linearly and semi-logarithmically (inset), with a DMSO concentration of 2% in all cases. ( b ) Overexpression of Uba1 or ubiquitin reduces sensitivity of cells to the cytostatic/cytotoxic effects of the compounds that also have in vitro activity against PCNA ubiquitination and Uba1~ubiquitin thioester formation (Figs and ) but not those without activity (Figs and ). Data represent mean and SD for % cell survival relative to parallel controls (DMSO alone) for three independent experiments, with a DMSO concentration of 2% in all cases. Higher survival values following compound treatment between Uba1- or ubiquitin-overexpressing HEK 293 cells compared to the corresponding empty vector-transfected control cells after 24-h compound treatment are indicated with an asterisk ( p < 0.003 in all cases by unpaired two-tailed Student’s t -tests). Treatment concentrations were at the IC 50 values initially calculated for each compound shown in Table from the data in ( a ), as well as 1 μM and 2 μM for puromycin—denoted as “Puro (1)” and “Puro (2),” respectively—to evaluate effects of an unrelated cytotoxic agent. Note: EC, Cat, Myr, and DM all exhibited only very weak activity at 500 μM in the cell viability experiments in ( a ), and we were not able to test over high enough concentration range for proper determination of their IC 50 values. However, of these four compounds, we chose to still examine whether Uba1 and/or ubiquitin overexpression affected cell survival for Myr at 3 mM and DM at 650 μM (corresponding to their crudely guesstimated IC 50 values), respectively, because Myr and DM had in vitro activity (Figs and ), whereas EC and Cat did not (Figs and ). Western blots of lysates from cells transfected with empty vector control, FLAG-ubiquitin-expressing, or FLAG-Uba1-expressing vector probed with anti-FLAG antibody, then stripped and probed with an anti-DNA polymerase δ catalytic subunit antibody as a loading control. The graph at right shows that Uba1 or ubiquitin overexpression by themselves had no effect on cell number in the parallel control cells in DMSO alone (mean and SD for three independent experiments), expressed as resorufin product signal in arbitrary fluorescence units (AFU) resulting from the metabolic reduction of resazurin in the assay. OE = overexpression. ( c ) Representative Western blot showing inhibition of global ubiquitination by EGCG in HEK 293 cells. Cells were treated with EGCG at 250 μM and 500 μM (with DMSO alone as control and a DMSO concentration of 1% in all cases) for 30 min, then MG132 was added to 50 μM, and cells were incubated for another 30 min, for a total EGCG treatment time of 1 h. Equivalent loadings of total protein from whole-cell lysates were subjected to Western blot analysis with anti-ubiquitin antibody and then with anti-β-tubulin antibody as a housekeeping protein loading control.

Article Snippet: His-tagged human Uba1 in the pET3a bacterial expression vector (Addgene Cat#63571) was expressed in the E . coli strain BL21(DE3) and purified with Ni-NTA agarose beads (Macherey-Nagel).

Techniques: Over Expression, Resazurin Assay, Concentration Assay, In Vitro, Activity Assay, Plasmid Preparation, Transfection, Two Tailed Test, Western Blot, Expressing, Fluorescence, Inhibition, Incubation

Journal: Gene Expression

Article Title: The λ Holin Accumulates Beyond the Lethal Triggering Concentration Under Hyperexpression Conditions

doi:

Figure Lengend Snippet: BACTERIAL STRAINS, BACTERIOPHAGES, PLASMIDS, AND OLIGONUCLEOTIDES

Article Snippet: To eliminate these factors in the expression of 5, the entire 5 reading frame, beginning with the codon 1 AUG, was fused directly to the efficient ribosome binding site of the T7 ϕ10 capsid protein gene in the pET3a expression vector (Novagen), resulting in pET3S107 ( ).

Techniques: Clone Assay, Expressing, Plasmid Preparation, Binding Assay, Sequencing

Journal: Gene Expression

Article Title: The λ Holin Accumulates Beyond the Lethal Triggering Concentration Under Hyperexpression Conditions

doi:

Figure Lengend Snippet: λCE6 infection profiles of MC4100 harboring pET3a clones. A600 values plateau between 30 and 40 min postinfection in the vector control, pET3a (□), whereas both pET3S107 ( ) and pET3S105 (•) display gradual decreases in absorbance.

Article Snippet: To eliminate these factors in the expression of 5, the entire 5 reading frame, beginning with the codon 1 AUG, was fused directly to the efficient ribosome binding site of the T7 ϕ10 capsid protein gene in the pET3a expression vector (Novagen), resulting in pET3S107 ( ).

Techniques: Infection, Clone Assay, Plasmid Preparation

Journal: Gene Expression

Article Title: The λ Holin Accumulates Beyond the Lethal Triggering Concentration Under Hyperexpression Conditions

doi:

Figure Lengend Snippet: Coomassie blue-stained SDS-PAGE. (A) Visible bands for S105 and S107 (glycine buffer system). pET3 clones were induced, membranes were collected, and proteins were extracted with 1% Triton X-100 and resolved by glycine SDS-PAGE as described in Materials and Methods. Lane 1 contains molecular weight markers with masses denoted on the left. Lanes 2–4 correspond to vector, S107, and S105, respectively. (B) Comparison of amounts of S107 accumulated in pET3S107 and pBS107 inductions. Whole-cell samples were resolved by glycine SDS-PAGE and visualized with Coomassie blue. Lanes 1 and 4: induced vector samples for pET3a and pBSM13−, respectively; lane 2: pET3S107; lane 3: pBSS107. (C) Localization of hyperexpressed S105. Molecular mass is indicated on the left. Lanes 1, 3, 5, and 7: samples obtained from induction of BL21 (DE3) pET11a; lanes 2, 4, 6, and 8: induction of BL21 (DE3) pETHS105. Lanes 1 and 2 represent the pellets obtained from the low-speed centrifugation step to isolate inclusion bodies. Lanes 3 and 4 represent the supernatants (cytoplasmic and periplasmic fractions) from the 100,000 × g centrifugation step to isolate total membranes. Lanes 5 and 6 correspond to the Triton-magnesium-solubilized membrane pellets. Lanes 7 and 8 represent the delipidated, LPS-deficient, Triton-insoluble fraction, obtained by methanol/chloroform extraction of the pellet from the Triton solubilization procedure. The arrow indicates the position of S105 (seen predominantly in lane 6).

Article Snippet: To eliminate these factors in the expression of 5, the entire 5 reading frame, beginning with the codon 1 AUG, was fused directly to the efficient ribosome binding site of the T7 ϕ10 capsid protein gene in the pET3a expression vector (Novagen), resulting in pET3S107 ( ).

Techniques: Staining, SDS Page, Clone Assay, Molecular Weight, Plasmid Preparation, Centrifugation