Review

pet3a expression vector (Thermo Fisher)

Thermo Fisher is a verified supplier

Thermo Fisher manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Thermo Fisher pet3a expression vector
Pet3a Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pet3a+expression+vector/us11684658-559-8-11
Average 86 stars, based on 1 article reviews

pet3a expression vector - by Bioz Stars, 2026-11

86/100 stars

Images

Expressing:

Article Title: Promiscuous Interactions of gp78 E3 ligase CUE domain with polyubiquitin chains
Article Snippet: .. gp78CUE expression and purification The CUE (aa: 453–504) of gp78 was subcloned into pET3a vector between Ndel and BamHi restriction sites from the template of full-length gp78 clone (gp78FL/pGEX). gp78CUE/PET3a gene construct was transformed to BL21 Star E. coli (Invitrogen). ..

Article Title: Vaccine adjuvants and formulations
Article Snippet: Murine α-lactalbumin DNA was inserted into the pET3a expression vector, (GeneArt) to provide a recombinant mouse α-lactalbumin containing an N-terminal FLAG-tag and a C-terminal 6×His-tag (FLAG-N-mαlac-C-HIS). .. Human α-lactalbumin DNA was also inserted into the pET3a expression vector (GeneArt) to provide a recombinant human α-lactalbumin containing a 6×His-tag linked to the N-terminus with the tobacco etch virus nuclear-inclusion-a endopeptidase (TEV protease) that serves as a cleavage site for removing the 6×His-tag from the recombinant human α-lactalbumin protein (HISTEV-N-hαlac-COOH). .. Plasmids containing these inserts were transformed in E. coli strain BL21 Star (Invitrogen, Carlsbad, Calif.).

Article Title: A bioengineered arginine-depleting enzyme as a long-lasting therapeutic agent against cancer.
Article Snippet: L-Arginine (L-Arg) depletion has attracted great attention in cancer therapy.. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production.. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product.

Article Title: Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer
Article Snippet: .. Expression plasmid pET3a was obtained from Invitrogen (San Diego, CA, USA). .. Human lung cancer cell lines A549, DMS114, NCI-H23 and NCI-H460 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Purification:

Plasmid Preparation:

Article Title: Structural studies of MMP-3 interaction with triple-helical collagen introduce the enzyme’s new roles in tissue remodelling
Article Snippet: .. They were overexpressed from a pET3a vector in E. coli BL21 (DE3) strain (Invitrogen). .. Transformed cells were grown to OD600 ~ 0.4, then induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and harvested after 4 h. Inclusion bodies were collected by lysing the cells in 0.05 M Tris-HCl pH 8, 0.1 M NaCl, 1 mM ethylenediaminotetraacetic acid (EDTA), 0.26 mg/ml lysozyme (Sigma-Aldrich), 0.5% Triton-X100, and dissolved in 8 M Urea, 50 μM ZnCl 2 , 20 mM Tris-HCl, pH 8.6, 20 mM dithiothreitol (DTT).

Article Title: Structural studies of the MMP-3 interaction with triple-helical collagen introduce new roles for the enzyme in tissue remodelling
Article Snippet: .. They were overexpressed from a pET3a vector in E. coli BL21 (DE3) strain (Invitrogen). .. Transformed cells were grown to OD600 ~ 0.4, then induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and harvested after 4 h. Inclusion bodies were collected by lysing the cells in 0.05 M Tris-HCl pH 8, 0.1 M NaCl, 1 mM ethylenediaminotetraacetic acid (EDTA), 0.26 mg/ml lysozyme (Sigma-Aldrich), 0.5% Triton-X100, and dissolved in 8 M Urea, 50 μM ZnCl 2 , 20 mM Tris-HCl, pH 8.6, 20 mM dithiothreitol (DTT).

Construct:

Transformation Assay:

Clone Assay:

Article Title: The sirtuin SIRT6 regulates stress granule formation in C. elegans and mammals.
Article Snippet: .. Generation of cDNA encoding sir-2.4 or K08F4.2 A cDNA encoding sir-2.4 was cloned into pET3a vector (Invitrogen) or L4440 vector (Addgene) after RT-PCR amplification from total RNA of wild-type C. elegans using Superscript II polymerase (Promega) according to the manufacturer’s instructions. ..

Reverse Transcription Polymerase Chain Reaction:

Amplification:

Recombinant:

Virus:

other:

Article Title: Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands
Article Snippet: In turn, phenyl HSL derivatives R6 and Q9 were very weak QscR agonists, with EC 50 values > 70 nM. table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Description Reference Strain E. coli JLD271 K-12 ΔlacX74 sdiA271::Cam; Cl R ( Lindsay and Ahmer, 2005 ) E. coli BL21 (DE3)pLysS F − omp T hsd SB (rB − , mB − ) gal dcm (DE3) pLysS(Cam R ) Invitrogen Plasmid pET3a-qscR Expression plasmid for full-length QscR ( Oinuma and Greenberg, 2011 ) pJN105Q arabinose-inducible QscR expression vector; Gm R ( Lee et al., 2006 ) pSC11-Q PA1897’-lacZ transcriptional fusion; QscR reporter vector; Ap R ( Eibergen et al., 2015 ) pJN105L arabinose-inducible LasR expression vector;Gm R ( Lee et al., 2006 ) pSC11 lasI’-lacZ transcriptional fusion; LasR reporter vector; Ap R ( Chugani et al., 2001 ) Open in a separate window Bacterial strains and plasmids used for in vivo assays table ft1 table-wrap mode="anchored" t5 caption a7 Compound IC 50 [μM] 95% CI a [μM] Inhibition (%) EC 50 μM] 95% CI [μM] Activation (%) 3OC12-AHL – – – 0.015 0.012 - 0.018 100 C12-AHL – – – 0.015 0.011 - 0.020 112 S3 – – – 0.036 0.021 - 0.060 88 CL – – – 0.016 0.010 - 0.027 86 Q9 0.026 0.015 - 0.042 80 0.12 0.0037 - 3.6 11 R6 0.042 0.017 - 0.10 58 0.074 0.025 - 0.22 23 Open in a separate window a CI = Confidence interval.

Image Search Results

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.

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

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.

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.

Techniques: Concentration Assay, Inhibition, Over Expression

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.

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.

Techniques: Activity Assay, Inhibition, Over Expression

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.

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.

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

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.

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.

Techniques:

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.

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.

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

BACTERIAL STRAINS, BACTERIOPHAGES, PLASMIDS, AND OLIGONUCLEOTIDES

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

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.

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.

Techniques: Infection, Clone Assay, Plasmid Preparation

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

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).

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

Review

pet3a expression vector (Thermo Fisher)

Structured Review

Images

Related Articles

Expressing:

Purification:

Plasmid Preparation:

Construct:

Transformation Assay:

Clone Assay:

Reverse Transcription Polymerase Chain Reaction:

Amplification:

Recombinant:

Virus:

other:

Similar Products

Image Search Results