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  • 99
    New England Biolabs e coli bl21 de3
    E Coli Bl21 De3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC e coli bl21 de3
    E Coli Bl21 De3, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bl21 de3 e coli
    Bl21 De3 E Coli, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e coli bl21 de3
    Recombinant AfIlv3A expression, purification and DHAD activity. A. Expression of recombinant Ilv3A protein. E. coli <t>BL21</t> <t>DE3</t> cells were transformed with pET30_Ilv3A, grown until OD600 > 0.5 and then incubated in the presence and absence of 0.5 mM IPTG for 20 h at 20°C. Ilv3A was purified from the bacterial lysate by immobilized metal ion affinity chromatography (IMAC). Protein fractions were separated by polyacrylamide gel electrophoresis on 4–12% Bis-Tris NuPage gels (Invitrogen) followed by staining with GelCode Blue staining reagent (Pierce). The molecular weights in kilo Daltons of marker proteins are indicated. Lane 1, E. coli cell lysate; lane 2, E. coli cell lysate following IPTG induction; lane 3, Ilv3A protein purified by IMAC. B. Dihydroxyacid dehydratase activity of recombinant Ilv3A. 2 µg Ilv3A was assayed in the presence of 0–60 mM L-threonic acid. Initial reaction velocities were calculated and plotted versus substrate concentration.
    E Coli Bl21 De3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs e coli bl21
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli <t>BL21</t> expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
    E Coli Bl21, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene e coli bl21 de3
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli <t>BL21</t> expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
    E Coli Bl21 De3, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore escherichia coli e coli bl21 de3 plyss
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli <t>BL21</t> expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
    Escherichia Coli E Coli Bl21 De3 Plyss, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bl21 de3 escherichia coli
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli <t>BL21</t> expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
    Bl21 De3 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beijing TransGen Biotech escherichia coli bl21 de3
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli <t>BL21</t> expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
    Escherichia Coli Bl21 De3, supplied by Beijing TransGen Biotech, used in various techniques. Bioz Stars score: 93/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies escherichia coli bl21 de3
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli <t>BL21</t> expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
    Escherichia Coli Bl21 De3, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co e coli bl21 de3
    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli <t>BL21</t> expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.
    E Coli Bl21 De3, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co escherichia coli bl21 de3
    SDS-PAGE and western blotting of S. scabiei PTK. Lanes: M, protein molecular weight markers (in kDa); 1, non-purified recombinant PTK (inclusion bodies from <t>Escherichia</t> coli <t>BL21</t> (DE3) expressing the protein); 2, purified recombinant PTK; 3, total crude proteins from S. scabiei ; 4, purified recombinant SsPTK detected in serum (diluted 1:100 with 0.01 M PBS) from a rabbit naturally infested with S. scabiei (experimental group); 5, purified recombinant SsPTK detected in rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS; positive control); 6, purified recombinant SsPTK detected in naïve rabbit serum (diluted 1:100 with 0.01 M PBS; negative control); 7, total crude proteins detected with rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS). Samples derived from the same experiment and gels/blots were processed in parallel. Cropping was used and full-length blots/gels are presented in supplementary information Figure S1 .
    Escherichia Coli Bl21 De3, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 91/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega e coli bl21 de3
    SDS-PAGE and western blotting of S. scabiei PTK. Lanes: M, protein molecular weight markers (in kDa); 1, non-purified recombinant PTK (inclusion bodies from <t>Escherichia</t> coli <t>BL21</t> (DE3) expressing the protein); 2, purified recombinant PTK; 3, total crude proteins from S. scabiei ; 4, purified recombinant SsPTK detected in serum (diluted 1:100 with 0.01 M PBS) from a rabbit naturally infested with S. scabiei (experimental group); 5, purified recombinant SsPTK detected in rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS; positive control); 6, purified recombinant SsPTK detected in naïve rabbit serum (diluted 1:100 with 0.01 M PBS; negative control); 7, total crude proteins detected with rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS). Samples derived from the same experiment and gels/blots were processed in parallel. Cropping was used and full-length blots/gels are presented in supplementary information Figure S1 .
    E Coli Bl21 De3, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Recombinant AfIlv3A expression, purification and DHAD activity. A. Expression of recombinant Ilv3A protein. E. coli BL21 DE3 cells were transformed with pET30_Ilv3A, grown until OD600 > 0.5 and then incubated in the presence and absence of 0.5 mM IPTG for 20 h at 20°C. Ilv3A was purified from the bacterial lysate by immobilized metal ion affinity chromatography (IMAC). Protein fractions were separated by polyacrylamide gel electrophoresis on 4–12% Bis-Tris NuPage gels (Invitrogen) followed by staining with GelCode Blue staining reagent (Pierce). The molecular weights in kilo Daltons of marker proteins are indicated. Lane 1, E. coli cell lysate; lane 2, E. coli cell lysate following IPTG induction; lane 3, Ilv3A protein purified by IMAC. B. Dihydroxyacid dehydratase activity of recombinant Ilv3A. 2 µg Ilv3A was assayed in the presence of 0–60 mM L-threonic acid. Initial reaction velocities were calculated and plotted versus substrate concentration.

    Journal: PLoS ONE

    Article Title: The Aspergillus fumigatus Dihydroxyacid Dehydratase Ilv3A/IlvC Is Required for Full Virulence

    doi: 10.1371/journal.pone.0043559

    Figure Lengend Snippet: Recombinant AfIlv3A expression, purification and DHAD activity. A. Expression of recombinant Ilv3A protein. E. coli BL21 DE3 cells were transformed with pET30_Ilv3A, grown until OD600 > 0.5 and then incubated in the presence and absence of 0.5 mM IPTG for 20 h at 20°C. Ilv3A was purified from the bacterial lysate by immobilized metal ion affinity chromatography (IMAC). Protein fractions were separated by polyacrylamide gel electrophoresis on 4–12% Bis-Tris NuPage gels (Invitrogen) followed by staining with GelCode Blue staining reagent (Pierce). The molecular weights in kilo Daltons of marker proteins are indicated. Lane 1, E. coli cell lysate; lane 2, E. coli cell lysate following IPTG induction; lane 3, Ilv3A protein purified by IMAC. B. Dihydroxyacid dehydratase activity of recombinant Ilv3A. 2 µg Ilv3A was assayed in the presence of 0–60 mM L-threonic acid. Initial reaction velocities were calculated and plotted versus substrate concentration.

    Article Snippet: Sequencing confirmed that the resulting vector, pET30_Ilv3A, was correctly constructed. pET30_Ilv3A was transformed into E. coli BL21 DE3 (Novagen) and expression was induced by IPTG as directed in the manufacturers protocol.

    Techniques: Recombinant, Expressing, Purification, Activity Assay, Transformation Assay, Incubation, Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Staining, Marker, Concentration Assay

    Overexpression of the recombinant T. pallidum Gpd and analysis of anti-Gpd immunoreactivity. (A) Coomassie blue-stained SDS-PAGE analysis of E. coli BL21 (DE3) pLysS expressing either the Gpd-pET-3a construct (lane 1, crude lysate; lane 2, soluble fraction; lane 3, insoluble fraction) or the pET-3a vector alone (lane 4, insoluble fraction). (B) Immunoblot analysis of anti-Gpd immunoreactivity on purified inclusion bodies from E. coli BL21 (DE3) pLysS expressing the Gpd-pET-3a construct. Lanes: 1, anti-Gpd polyclonal antiserum, 2, E. coli -adsorbed anti-Gpd polyclonal antiserum. Each lane contains approximately 2 μg of total bacterial lysate and soluble or insoluble bacterial fractions, and molecular mass standards in kilodaltons are indicated at the left of each panel. In each panel, the recombinant T. pallidum Gpd is indicated by an arrow.

    Journal: Infection and Immunity

    Article Title: Function and Protective Capacity of Treponema pallidum subsp. pallidum Glycerophosphodiester Phosphodiesterase

    doi:

    Figure Lengend Snippet: Overexpression of the recombinant T. pallidum Gpd and analysis of anti-Gpd immunoreactivity. (A) Coomassie blue-stained SDS-PAGE analysis of E. coli BL21 (DE3) pLysS expressing either the Gpd-pET-3a construct (lane 1, crude lysate; lane 2, soluble fraction; lane 3, insoluble fraction) or the pET-3a vector alone (lane 4, insoluble fraction). (B) Immunoblot analysis of anti-Gpd immunoreactivity on purified inclusion bodies from E. coli BL21 (DE3) pLysS expressing the Gpd-pET-3a construct. Lanes: 1, anti-Gpd polyclonal antiserum, 2, E. coli -adsorbed anti-Gpd polyclonal antiserum. Each lane contains approximately 2 μg of total bacterial lysate and soluble or insoluble bacterial fractions, and molecular mass standards in kilodaltons are indicated at the left of each panel. In each panel, the recombinant T. pallidum Gpd is indicated by an arrow.

    Article Snippet: Anti-Gpd polyclonal antiserum was raised in a New Zealand White rabbit by immunizing three times with 100 μg of each of the inclusion bodies purified from E. coli BL21 (DE3) pLysS transformed with the Gpd-pET-3a construct, emulsified in the Ribi adjuvant MPL + TDM + CWS (monophosphoryl lipid A + trehalose dicorynomycolate + cell wall skeleton) (Sigma).

    Techniques: Over Expression, Recombinant, Staining, SDS Page, Expressing, Positron Emission Tomography, Construct, Plasmid Preparation, Purification

    Immunoblot analyses of the Ig-binding capability of recombinant T. pallidum Gpd. Shown are inclusion bodies purified from E. coli BL21 (DE3) pLysS transformed with either the pET-3a-Gpd construct (lanes 1, 3, 5, and 7) or with the pET-3a vector alone (lanes 2, 4, 6, and 8). Molecular mass standards in kilodaltons are indicated on the left of each panel. (A) Specificity of recombinant T. pallidum Gpd for binding various Ig classes. Lanes: 1 and 2, human IgA and goat F(ab′) 2 anti-human IgA (alpha chain specific); 3 and 4, human IgD and goat F(ab′) 2 anti-human IgD (delta chain specific); 5 and 6, human IgG and goat F(ab′) 2 anti-human IgG (gamma chain specific); 7 and 8, human IgM and goat F(ab′) 2 anti-human IgM (mu chain specific). (B) Investigation of the binding specificity of recombinant T. pallidum Gpd for human IgG. In all cases the secondary antibody used was goat F(ab′) 2 anti-human IgG (gamma chain specific). Lanes: 1 and 2, intact human IgG; 3 and 4, human IgG Fc fragment; 5 and 6, human IgG Fab fragment; 7 and 8, no primary Ig.

    Journal: Infection and Immunity

    Article Title: Function and Protective Capacity of Treponema pallidum subsp. pallidum Glycerophosphodiester Phosphodiesterase

    doi:

    Figure Lengend Snippet: Immunoblot analyses of the Ig-binding capability of recombinant T. pallidum Gpd. Shown are inclusion bodies purified from E. coli BL21 (DE3) pLysS transformed with either the pET-3a-Gpd construct (lanes 1, 3, 5, and 7) or with the pET-3a vector alone (lanes 2, 4, 6, and 8). Molecular mass standards in kilodaltons are indicated on the left of each panel. (A) Specificity of recombinant T. pallidum Gpd for binding various Ig classes. Lanes: 1 and 2, human IgA and goat F(ab′) 2 anti-human IgA (alpha chain specific); 3 and 4, human IgD and goat F(ab′) 2 anti-human IgD (delta chain specific); 5 and 6, human IgG and goat F(ab′) 2 anti-human IgG (gamma chain specific); 7 and 8, human IgM and goat F(ab′) 2 anti-human IgM (mu chain specific). (B) Investigation of the binding specificity of recombinant T. pallidum Gpd for human IgG. In all cases the secondary antibody used was goat F(ab′) 2 anti-human IgG (gamma chain specific). Lanes: 1 and 2, intact human IgG; 3 and 4, human IgG Fc fragment; 5 and 6, human IgG Fab fragment; 7 and 8, no primary Ig.

    Article Snippet: Anti-Gpd polyclonal antiserum was raised in a New Zealand White rabbit by immunizing three times with 100 μg of each of the inclusion bodies purified from E. coli BL21 (DE3) pLysS transformed with the Gpd-pET-3a construct, emulsified in the Ribi adjuvant MPL + TDM + CWS (monophosphoryl lipid A + trehalose dicorynomycolate + cell wall skeleton) (Sigma).

    Techniques: Binding Assay, Recombinant, Purification, Transformation Assay, Positron Emission Tomography, Construct, Plasmid Preparation

    Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli BL21 expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.

    Journal: Frontiers in Microbiology

    Article Title: Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation

    doi: 10.3389/fmicb.2018.00642

    Figure Lengend Snippet: Importance of auto-processing for oligomerization and caseinolytic activity of HtrA Hp expressed in E. coli . The amino-terminus of HtrA Hp was mutagenized by generating deletion variants and expressed as GST-tagged variants in E. coli ). E. coli BL21 expressing GST-tagged HtrA Hp without the signal peptide (ΔSP) and empty vector E. coli BL21 were used as controls. (A) After induction of protein expression by IPTG, the protein lysates were subjected to Coomassie staining. Overexpression of the HtrA Hp p55 monomers and the GST-tagged variants was observed (arrows). The amino-terminal deletion variants (ΔN1 and ΔN2, yellow asterisks) exhibit a lower molecular weight compared to HtrA Hp ΔSP (black asterisk). (B) The bacterial lysates were subjected to immunoblotting against HtrA Hp and GST. Overexpression of the GST-tagged HtrA Hp p55 variants was confirmed by detecting the HtrA Hp or GST protein, respectively (arrows). In addition, immunoblotting against HtrA Hp showed the presence of HtrA Hp monomers without GST-tag (arrow). The p52 form is migrating slightly below p55 (red asterisks). (C) Finally, the bacterial lysates were analyzed by casein zymography. For wt HtrA Hp ΔSP, a strong caseinolytic activity of the HtrA Hp monomers and its GST-tagged variants was observed (arrows). The p52 form is migrating slightly below p55 (red asterisks). Moreover, caseinolytic activity for the HtrA Hp trimer and its GST-tagged variant was shown (arrows). The ΔN1 and ΔN2 variants revealed no proteolytic activity (yellow asterisks). All assays were done in triplicates.

    Article Snippet: To further characterize if the reduction of activity resulted from loss of the entire amino-terminus, we created HtrAHp variants without the amino-terminus including cleavage site H46/D47 (ΔN1) or H46/D47 and K50/D51 (ΔN2), respectively, and expressed the HtrAHp variants heterologously in E. coli BL21.

    Techniques: Activity Assay, Expressing, Plasmid Preparation, Staining, Over Expression, Molecular Weight, Zymography, Variant Assay

    SDS-PAGE and western blotting of S. scabiei PTK. Lanes: M, protein molecular weight markers (in kDa); 1, non-purified recombinant PTK (inclusion bodies from Escherichia coli BL21 (DE3) expressing the protein); 2, purified recombinant PTK; 3, total crude proteins from S. scabiei ; 4, purified recombinant SsPTK detected in serum (diluted 1:100 with 0.01 M PBS) from a rabbit naturally infested with S. scabiei (experimental group); 5, purified recombinant SsPTK detected in rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS; positive control); 6, purified recombinant SsPTK detected in naïve rabbit serum (diluted 1:100 with 0.01 M PBS; negative control); 7, total crude proteins detected with rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS). Samples derived from the same experiment and gels/blots were processed in parallel. Cropping was used and full-length blots/gels are presented in supplementary information Figure S1 .

    Journal: Scientific Reports

    Article Title: Expression and characterisation of a Sarcoptes scabiei protein tyrosine kinase as a potential antigen for scabies diagnosis

    doi: 10.1038/s41598-017-10326-w

    Figure Lengend Snippet: SDS-PAGE and western blotting of S. scabiei PTK. Lanes: M, protein molecular weight markers (in kDa); 1, non-purified recombinant PTK (inclusion bodies from Escherichia coli BL21 (DE3) expressing the protein); 2, purified recombinant PTK; 3, total crude proteins from S. scabiei ; 4, purified recombinant SsPTK detected in serum (diluted 1:100 with 0.01 M PBS) from a rabbit naturally infested with S. scabiei (experimental group); 5, purified recombinant SsPTK detected in rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS; positive control); 6, purified recombinant SsPTK detected in naïve rabbit serum (diluted 1:100 with 0.01 M PBS; negative control); 7, total crude proteins detected with rabbit anti-PTK serum (diluted 1:100 with 0.01 M PBS). Samples derived from the same experiment and gels/blots were processed in parallel. Cropping was used and full-length blots/gels are presented in supplementary information Figure S1 .

    Article Snippet: The resulting fragment was cloned into the pET32a(+) expression vector (Invitrogen, Beijing, China), and the construct was transformed into Escherichia coli BL21 (DE3) (TIANGEN, Beijing, China) for protein expression which was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at 37 °C for 10 h. Recombinant SsPTK protein was purified from inclusion bodies in denaturing conditions (8 M urea) by chromatography using a Ni-NTA His-tag affinity kit (Bio-Rad, USA) according to the manufacturer’s instructions.

    Techniques: SDS Page, Western Blot, Molecular Weight, Purification, Recombinant, Expressing, Positive Control, Negative Control, Derivative Assay