recombinant hphtra proteins Search Results


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  • 86
    GE Healthcare recombinant gst
    Recombinant Gst, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    94
    R&D Systems recombinant e cadherin
    Recombinant E Cadherin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant e cadherin/product/R&D Systems
    Average 94 stars, based on 1 article reviews
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    86
    Millipore hepes buffer
    A novel FRET peptide assay to detect H. pylori HtrA activity. ( A ) The cleavage specificity profile of HpHtrA obtained from DIPPS profiling is represented as an iceLogo, with significantly enriched and under-represented amino acids above and below the x -axis, respectively. The scissile peptide bond between P1 and P1′ is shown as a gray dashed line in the iceLogo. The model on the right represents the domain structure of human E-cadherin (hCdh1), which is composed of the extracellular domain (EC1–EC5), a transmembrane domain (TMD) and an intracellular domain (IC). HtrA cleavage sites have been identified (red arrows) in the Ca ++ -binding sites located between the individual EC regions. According to the iceLogo, an additional cleavage site for HtrA is present in the linker region between the EC5 domain and TMD. ( B ) The sequence AQRVAF harboring 2-aminobenzoyl (2-Abz) as fluorophore and 3-nitro-tyrosine Y(NO 2 ) as a quencher is hydrolyzed by trypsin with arginine (R) at position P1 and by HpHtrA with valine (V) at position P1. ( C ) 5 µM of the FRET peptide were incubated with 250 nM of HpHtrA wild type (wt), its isogenic inactive mutant (SA), or 125 nM trypsin for 180 min in 50 mM <t>HEPES</t> <t>buffer</t> (pH 7.4) at 37 °C. The data represent the relative fluorescent units (RFU) ± S.D. with the fluorescent signal obtained from trypsin-treated FRET peptide set as 100%. Asterisks indicate statistically significant differences (**** p < 0.0001). ( D ) 4 µM of the FRET peptide were incubated with indicated concentrations of HpHtrA wt or SA for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the RFU ± S.D. with the fluorescent signals obtained from FRET peptide treated with 400 nM HpHtrA wt for 180 min set as 100%.
    Hepes Buffer, supplied by Millipore, 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/result/hepes buffer/product/Millipore
    Average 86 stars, based on 1 article reviews
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    hepes buffer - by Bioz Stars, 2023-02
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    86
    Millipore well pcr plate
    A novel FRET peptide assay to detect H. pylori HtrA activity. ( A ) The cleavage specificity profile of HpHtrA obtained from DIPPS profiling is represented as an iceLogo, with significantly enriched and under-represented amino acids above and below the x -axis, respectively. The scissile peptide bond between P1 and P1′ is shown as a gray dashed line in the iceLogo. The model on the right represents the domain structure of human E-cadherin (hCdh1), which is composed of the extracellular domain (EC1–EC5), a transmembrane domain (TMD) and an intracellular domain (IC). HtrA cleavage sites have been identified (red arrows) in the Ca ++ -binding sites located between the individual EC regions. According to the iceLogo, an additional cleavage site for HtrA is present in the linker region between the EC5 domain and TMD. ( B ) The sequence AQRVAF harboring 2-aminobenzoyl (2-Abz) as fluorophore and 3-nitro-tyrosine Y(NO 2 ) as a quencher is hydrolyzed by trypsin with arginine (R) at position P1 and by HpHtrA with valine (V) at position P1. ( C ) 5 µM of the FRET peptide were incubated with 250 nM of HpHtrA wild type (wt), its isogenic inactive mutant (SA), or 125 nM trypsin for 180 min in 50 mM <t>HEPES</t> <t>buffer</t> (pH 7.4) at 37 °C. The data represent the relative fluorescent units (RFU) ± S.D. with the fluorescent signal obtained from trypsin-treated FRET peptide set as 100%. Asterisks indicate statistically significant differences (**** p < 0.0001). ( D ) 4 µM of the FRET peptide were incubated with indicated concentrations of HpHtrA wt or SA for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the RFU ± S.D. with the fluorescent signals obtained from FRET peptide treated with 400 nM HpHtrA wt for 180 min set as 100%.
    Well Pcr Plate, supplied by Millipore, 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/result/well pcr plate/product/Millipore
    Average 86 stars, based on 1 article reviews
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    well pcr plate - by Bioz Stars, 2023-02
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    99
    GE Healthcare glutathione sepharose beads
    A novel FRET peptide assay to detect H. pylori HtrA activity. ( A ) The cleavage specificity profile of HpHtrA obtained from DIPPS profiling is represented as an iceLogo, with significantly enriched and under-represented amino acids above and below the x -axis, respectively. The scissile peptide bond between P1 and P1′ is shown as a gray dashed line in the iceLogo. The model on the right represents the domain structure of human E-cadherin (hCdh1), which is composed of the extracellular domain (EC1–EC5), a transmembrane domain (TMD) and an intracellular domain (IC). HtrA cleavage sites have been identified (red arrows) in the Ca ++ -binding sites located between the individual EC regions. According to the iceLogo, an additional cleavage site for HtrA is present in the linker region between the EC5 domain and TMD. ( B ) The sequence AQRVAF harboring 2-aminobenzoyl (2-Abz) as fluorophore and 3-nitro-tyrosine Y(NO 2 ) as a quencher is hydrolyzed by trypsin with arginine (R) at position P1 and by HpHtrA with valine (V) at position P1. ( C ) 5 µM of the FRET peptide were incubated with 250 nM of HpHtrA wild type (wt), its isogenic inactive mutant (SA), or 125 nM trypsin for 180 min in 50 mM <t>HEPES</t> <t>buffer</t> (pH 7.4) at 37 °C. The data represent the relative fluorescent units (RFU) ± S.D. with the fluorescent signal obtained from trypsin-treated FRET peptide set as 100%. Asterisks indicate statistically significant differences (**** p < 0.0001). ( D ) 4 µM of the FRET peptide were incubated with indicated concentrations of HpHtrA wt or SA for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the RFU ± S.D. with the fluorescent signals obtained from FRET peptide treated with 400 nM HpHtrA wt for 180 min set as 100%.
    Glutathione Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione sepharose beads/product/GE Healthcare
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glutathione sepharose beads - by Bioz Stars, 2023-02
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    Image Search Results


    A novel FRET peptide assay to detect H. pylori HtrA activity. ( A ) The cleavage specificity profile of HpHtrA obtained from DIPPS profiling is represented as an iceLogo, with significantly enriched and under-represented amino acids above and below the x -axis, respectively. The scissile peptide bond between P1 and P1′ is shown as a gray dashed line in the iceLogo. The model on the right represents the domain structure of human E-cadherin (hCdh1), which is composed of the extracellular domain (EC1–EC5), a transmembrane domain (TMD) and an intracellular domain (IC). HtrA cleavage sites have been identified (red arrows) in the Ca ++ -binding sites located between the individual EC regions. According to the iceLogo, an additional cleavage site for HtrA is present in the linker region between the EC5 domain and TMD. ( B ) The sequence AQRVAF harboring 2-aminobenzoyl (2-Abz) as fluorophore and 3-nitro-tyrosine Y(NO 2 ) as a quencher is hydrolyzed by trypsin with arginine (R) at position P1 and by HpHtrA with valine (V) at position P1. ( C ) 5 µM of the FRET peptide were incubated with 250 nM of HpHtrA wild type (wt), its isogenic inactive mutant (SA), or 125 nM trypsin for 180 min in 50 mM HEPES buffer (pH 7.4) at 37 °C. The data represent the relative fluorescent units (RFU) ± S.D. with the fluorescent signal obtained from trypsin-treated FRET peptide set as 100%. Asterisks indicate statistically significant differences (**** p < 0.0001). ( D ) 4 µM of the FRET peptide were incubated with indicated concentrations of HpHtrA wt or SA for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the RFU ± S.D. with the fluorescent signals obtained from FRET peptide treated with 400 nM HpHtrA wt for 180 min set as 100%.

    Journal: Scientific Reports

    Article Title: A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

    doi: 10.1038/s41598-020-67578-2

    Figure Lengend Snippet: A novel FRET peptide assay to detect H. pylori HtrA activity. ( A ) The cleavage specificity profile of HpHtrA obtained from DIPPS profiling is represented as an iceLogo, with significantly enriched and under-represented amino acids above and below the x -axis, respectively. The scissile peptide bond between P1 and P1′ is shown as a gray dashed line in the iceLogo. The model on the right represents the domain structure of human E-cadherin (hCdh1), which is composed of the extracellular domain (EC1–EC5), a transmembrane domain (TMD) and an intracellular domain (IC). HtrA cleavage sites have been identified (red arrows) in the Ca ++ -binding sites located between the individual EC regions. According to the iceLogo, an additional cleavage site for HtrA is present in the linker region between the EC5 domain and TMD. ( B ) The sequence AQRVAF harboring 2-aminobenzoyl (2-Abz) as fluorophore and 3-nitro-tyrosine Y(NO 2 ) as a quencher is hydrolyzed by trypsin with arginine (R) at position P1 and by HpHtrA with valine (V) at position P1. ( C ) 5 µM of the FRET peptide were incubated with 250 nM of HpHtrA wild type (wt), its isogenic inactive mutant (SA), or 125 nM trypsin for 180 min in 50 mM HEPES buffer (pH 7.4) at 37 °C. The data represent the relative fluorescent units (RFU) ± S.D. with the fluorescent signal obtained from trypsin-treated FRET peptide set as 100%. Asterisks indicate statistically significant differences (**** p < 0.0001). ( D ) 4 µM of the FRET peptide were incubated with indicated concentrations of HpHtrA wt or SA for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the RFU ± S.D. with the fluorescent signals obtained from FRET peptide treated with 400 nM HpHtrA wt for 180 min set as 100%.

    Article Snippet: Recombinant HpHtrA proteins (wt, S 164 A, D 165 A, S 166 A, D 168 A) in 50 mM HEPES buffer (pH 7.4) were mixed with SYPRO Orange (Sigma-Aldrich, Vienna, Austria) to final concentrations of 4 µM HpHtrA and 7 × SYPRO Orange in a 96-well PCR plate.

    Techniques: Activity Assay, Binding Assay, Sequencing, Incubation, Mutagenesis

    Divalent cations modulate the activity of HpHtrA. ( A ) 10 µg casein composed of α S1 -, α S2 - and β-casein were incubated with 250 ng HpHtrA for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Where indicated, 1 mM of the different divalent ions, EDTA, or EGTA was added. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue G250. ( B ) 50 ng hCdh1 were incubated with 250 ng HpHtrA for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Where indicated, 1 mM of the different divalent ions, EDTA, or EGTA was added. Full length hCdh1 (Cdh1 FL , 125 kDa) and hCdh1 cleavage fragments were detected by Western blot using an antibody recognizing the EC5 domain of hCdh1. HpHtrA and the auto-processed HpHtrA (HpHtrAs) were detected using a polyclonal HpHtrA antibody.

    Journal: Scientific Reports

    Article Title: A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

    doi: 10.1038/s41598-020-67578-2

    Figure Lengend Snippet: Divalent cations modulate the activity of HpHtrA. ( A ) 10 µg casein composed of α S1 -, α S2 - and β-casein were incubated with 250 ng HpHtrA for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Where indicated, 1 mM of the different divalent ions, EDTA, or EGTA was added. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue G250. ( B ) 50 ng hCdh1 were incubated with 250 ng HpHtrA for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Where indicated, 1 mM of the different divalent ions, EDTA, or EGTA was added. Full length hCdh1 (Cdh1 FL , 125 kDa) and hCdh1 cleavage fragments were detected by Western blot using an antibody recognizing the EC5 domain of hCdh1. HpHtrA and the auto-processed HpHtrA (HpHtrAs) were detected using a polyclonal HpHtrA antibody.

    Article Snippet: Recombinant HpHtrA proteins (wt, S 164 A, D 165 A, S 166 A, D 168 A) in 50 mM HEPES buffer (pH 7.4) were mixed with SYPRO Orange (Sigma-Aldrich, Vienna, Austria) to final concentrations of 4 µM HpHtrA and 7 × SYPRO Orange in a 96-well PCR plate.

    Techniques: Activity Assay, Incubation, SDS Page, Staining, Western Blot

    ZnCl 2 and CuCl 2 inhibit HpHtrA activity in a concentration-dependent manner. ( A ) 5 µM FRET peptide were incubated with 250 nM HpHtrA and increasing concentrations of ZnCl 2 (left panel) or CuCl 2 (right panel) for 15 min (black bars) and 180 min (grey bars) at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the relative fluorescence units (RFU) ± S.D. with fluorescent signals obtained from FRET peptide treated with HpHtrA wt set as 100%. Asterisks indicate statistically significant differences (**** p < 0.0001; ns, non-significant). ( B ) 50 ng hCdh1 were incubated with 250 ng HpHtrA wt or inactive mutant (SA) and increasing concentrations of ZnCl 2 (left panel) or CuCl 2 (right panel) for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Full length hCdh1 (Cdh1 FL ) and cleavage fragments were detected by Western blot using an antibody recognizing the EC5 domain of hCdh1. HpHtrA and the auto-processed short HpHtrA (HpHtrAs) were detected using a polyclonal antibody. ( C ) 10 µg casein composed of α S1 -, α S2 - and β-casein were incubated with 250 ng HpHtrA wt or inactive mutant (SA) and with increasing concentrations of ZnCl 2 (left panel) or CuCl 2 (right panel). After incubation at 37 °C for 16 h proteins were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G250.

    Journal: Scientific Reports

    Article Title: A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

    doi: 10.1038/s41598-020-67578-2

    Figure Lengend Snippet: ZnCl 2 and CuCl 2 inhibit HpHtrA activity in a concentration-dependent manner. ( A ) 5 µM FRET peptide were incubated with 250 nM HpHtrA and increasing concentrations of ZnCl 2 (left panel) or CuCl 2 (right panel) for 15 min (black bars) and 180 min (grey bars) at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the relative fluorescence units (RFU) ± S.D. with fluorescent signals obtained from FRET peptide treated with HpHtrA wt set as 100%. Asterisks indicate statistically significant differences (**** p < 0.0001; ns, non-significant). ( B ) 50 ng hCdh1 were incubated with 250 ng HpHtrA wt or inactive mutant (SA) and increasing concentrations of ZnCl 2 (left panel) or CuCl 2 (right panel) for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Full length hCdh1 (Cdh1 FL ) and cleavage fragments were detected by Western blot using an antibody recognizing the EC5 domain of hCdh1. HpHtrA and the auto-processed short HpHtrA (HpHtrAs) were detected using a polyclonal antibody. ( C ) 10 µg casein composed of α S1 -, α S2 - and β-casein were incubated with 250 ng HpHtrA wt or inactive mutant (SA) and with increasing concentrations of ZnCl 2 (left panel) or CuCl 2 (right panel). After incubation at 37 °C for 16 h proteins were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G250.

    Article Snippet: Recombinant HpHtrA proteins (wt, S 164 A, D 165 A, S 166 A, D 168 A) in 50 mM HEPES buffer (pH 7.4) were mixed with SYPRO Orange (Sigma-Aldrich, Vienna, Austria) to final concentrations of 4 µM HpHtrA and 7 × SYPRO Orange in a 96-well PCR plate.

    Techniques: Activity Assay, Concentration Assay, Incubation, Fluorescence, Mutagenesis, Western Blot, SDS Page, Staining

    Proteolytic activity of HpHtrA loop mutants. To analyze the proteolytic activity of HtrA wt and its isogenic mutants HpHtrA S 164 A, HpHtrA D 165 A, HpHtrA S 166 A, HpHtrA D 168 A, and the inactive HpHtrA S 221 A, 10 µg casein ( A ) or 50 ng E-cadherin (hCdh1) ( B ) were incubated with 250 ng HtrA variants as indicated for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Cleavage of casein was analyzed by Coomassie-stained SDS-PAGE and hCdh1 cleavage was determined by Western blot analysis using an antibody recognizing the EC5 domain of hCdh1. HpHtrA and the auto-processed HpHtrAs were detected as indicated. ( C ) The activity of the ligand-binding loop mutants was analyzed using the FRET peptide as a substrate. 5 µM FRET peptide were incubated with 250 nM HpHtrA wt (black circle), HpHtrA S 164 A (red square), HpHtrA D 165 A (green triangle), HpHtrA S 166 A (yellow inverted triangles), and HpHtrA D 168 A (blue rhombus) for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the relative fluorescent units (RFU) ± S.D. with fluorescent signals obtained from FRET peptide treated with HpHtrA wt for 180 min set as 100%.

    Journal: Scientific Reports

    Article Title: A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

    doi: 10.1038/s41598-020-67578-2

    Figure Lengend Snippet: Proteolytic activity of HpHtrA loop mutants. To analyze the proteolytic activity of HtrA wt and its isogenic mutants HpHtrA S 164 A, HpHtrA D 165 A, HpHtrA S 166 A, HpHtrA D 168 A, and the inactive HpHtrA S 221 A, 10 µg casein ( A ) or 50 ng E-cadherin (hCdh1) ( B ) were incubated with 250 ng HtrA variants as indicated for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Cleavage of casein was analyzed by Coomassie-stained SDS-PAGE and hCdh1 cleavage was determined by Western blot analysis using an antibody recognizing the EC5 domain of hCdh1. HpHtrA and the auto-processed HpHtrAs were detected as indicated. ( C ) The activity of the ligand-binding loop mutants was analyzed using the FRET peptide as a substrate. 5 µM FRET peptide were incubated with 250 nM HpHtrA wt (black circle), HpHtrA S 164 A (red square), HpHtrA D 165 A (green triangle), HpHtrA S 166 A (yellow inverted triangles), and HpHtrA D 168 A (blue rhombus) for 180 min at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the relative fluorescent units (RFU) ± S.D. with fluorescent signals obtained from FRET peptide treated with HpHtrA wt for 180 min set as 100%.

    Article Snippet: Recombinant HpHtrA proteins (wt, S 164 A, D 165 A, S 166 A, D 168 A) in 50 mM HEPES buffer (pH 7.4) were mixed with SYPRO Orange (Sigma-Aldrich, Vienna, Austria) to final concentrations of 4 µM HpHtrA and 7 × SYPRO Orange in a 96-well PCR plate.

    Techniques: Activity Assay, Incubation, Staining, SDS Page, Western Blot, Ligand Binding Assay