Journal: Biochemistry and Biophysics Reports

Article Title: The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

doi: 10.1016/j.bbrep.2016.11.011

Figure Lengend Snippet: Aggregation of ADH and Hsp60 monitored by using light scattering. ADH (1 μM) or Hsp60 (1 μM) was incubated in 50 mM Tris-HCl (pH 7.5-6.5) or 50 mM sodium citrate (pH 6.0-5.0) at 25 °C. The figure shows the % aggregation of ADH (diagonal bars) and Hsp60 (open bars) relative to the maximal aggregation (100%) corresponding to the highest scattering signal detected for ADH (pH 5.5).

Article Snippet: The H. pylori Hsp60 gene was synthesized by GenScript (New Jersey) and inserted into the expression vector pET-22b(+).

Techniques: Incubation

Journal: Biochemistry and Biophysics Reports

Article Title: The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

doi: 10.1016/j.bbrep.2016.11.011

Figure Lengend Snippet: Time course of the light scattering of ADH. ADH (1 μM) was added after 5 min to 50 mM sodium citrate, pH 5.5 at 25 °C without (closed circles) or with 1 μM Hsp60 (open circles).

Article Snippet: The H. pylori Hsp60 gene was synthesized by GenScript (New Jersey) and inserted into the expression vector pET-22b(+).

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

doi: 10.1016/j.bbrep.2016.11.011

Figure Lengend Snippet: Suppression of aggregation of ADH by Hsp60 monitored by using light scattering. Hsp60 (1 μM) was incubated in 50 mM Tris-HCl (pH 7.5-6.5) or 50 mM sodium citrate (pH 6.0-5.0) at 25 °C. ADH (1 μM) was added after 5 min and light scattering was recorded for 30 min after the addition of ADH to the buffers.

Article Snippet: The H. pylori Hsp60 gene was synthesized by GenScript (New Jersey) and inserted into the expression vector pET-22b(+).

Techniques: Incubation

Journal: Biochemistry and Biophysics Reports

Article Title: The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

doi: 10.1016/j.bbrep.2016.11.011

Figure Lengend Snippet: Tryptophan fluorescence of the Y359W mutant of Hsp60. The Y359W mutant was incubated with 50 mM Tris-HCl, pH 7.5 or 50 mM sodium citrate pH 5.5 and fluorescence recorded as described in “Materials and methods”. The final concentration of the Hsp60 mutant was 2.0 μM (monomers).

Article Snippet: The H. pylori Hsp60 gene was synthesized by GenScript (New Jersey) and inserted into the expression vector pET-22b(+).

Techniques: Fluorescence, Mutagenesis, Incubation, Concentration Assay

Journal: Biochemistry and Biophysics Reports

Article Title: The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

doi: 10.1016/j.bbrep.2016.11.011

Figure Lengend Snippet: Measurement of exposure of hydrophobic surfaces of Hsp60. Fluorescence emission spectra were recorded for bisANS alone in 50 mM Tris-HCl, pH 7.5 or 50 mM sodium citrate, pH 5.5 (lowest spectra). Hsp60 was added to bisANS in 50 mM Tris-HCl, pH 7.5 (middle spectrum) or to bisANS in 50 mM sodium citrate, pH 5.5 (highest spectrum). The final concentrations of bisANS and Hsp60 were 10 μM and 1 μM (monomers), respectively.

Article Snippet: The H. pylori Hsp60 gene was synthesized by GenScript (New Jersey) and inserted into the expression vector pET-22b(+).

Techniques: Fluorescence

Journal: Biochemistry and Biophysics Reports

Article Title: The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

doi: 10.1016/j.bbrep.2016.11.011

Figure Lengend Snippet: Measurement of exposure of hydrophobic surfaces of ADH. Fluorescence emission spectra were recorded for bisANS alone in 50 mM Tris-HCl, pH 7.5 or 50 mM sodium citrate, pH 5.5 (lowest spectra). ADH was added to bisANS in 50 mM Tris-HCl, pH 7.5 (middle spectrum) or to bisANS in 50 mM sodium citrate, pH 5.5 (highest spectrum). The final concentrations of bisANS and Hsp60 were 10 μM and 1 μM (monomers), respectively.

Article Snippet: The H. pylori Hsp60 gene was synthesized by GenScript (New Jersey) and inserted into the expression vector pET-22b(+).

Techniques: Fluorescence

Journal: Cellular & molecular biology letters

Article Title: γ-2 and GSG1L bind with comparable affinities to the tetrameric GluA1 core.

doi: 10.1186/s11658-023-00470-9

Figure Lengend Snippet: Fig. 3 The anti-GFP nanobody binds GFP but not mNeonGreen. A A647-Nb injected into oocytes expressing GluA1-GFP shows strong colocalization with GluA1-GFP. B A647-Nb does not label the extracellular GFP of GFP-GluA1. Weak red spots in the background are from intracellular A647 molecules. C A647-Nb binds to the non-fluorescent GFP(Y66L) of GFP-GluA1-GFP(Y66L). D A647-Nb does not label intracellular mNeonGreen of GluA1-mNeonGreen. E Photobleaching steps of GluA1-mNeonGreen. F A representative distribution of mNeonGreen photobleaching steps with a binomial fit. Scale bars 2 µm

Article Snippet: For the nanobody expression vector pET22b-pelB-K4-vhhGFP4-6xHis, a linker containing four lysine residues for dye conjugation and the anti-GFP nanobody (Addgene #35579) were cloned into the pET22b vector (Novagen, EMD Millipore) (full sequence see Additional file 1: Note 3).

Techniques: Injection, Expressing

Journal: Pharmaceutics

Article Title: In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics

doi: 10.3390/pharmaceutics12121217

Figure Lengend Snippet: Characterization of the different protein formats employed in this study. ( A ) Schematic representation of multimeric fusion proteins. The presence of a cationic peptide (GWH1) in the N-terminus, plus the His6 tag in C-terminus, may induce the formation of protein-only nanoparticles (PNPs). A linker between GWH1 and the scaffold protein was added in order to increase flexibility and facilitate self-assembling process. ( B ) TGX Stain-free TM analysis showing the purity of the different protein designs in both, soluble proteins ( B1 ) and IBs ( B2 ). ( C ) Western blot showing the qualitative amount of protein released from the different IBs. ( D ) Evaluation of self-assembling capabilities of purified soluble proteins by size determination using DLS. PNPs: protein nanoparticles; UPs: unassembled proteins. ( E ) Size distribution of the protein samples obtained after solubilization from different IBs analyzed by DLS.

Article Snippet: All DNA sequences (GWH1-GFP, GWH1-IFN-γ, IFN-γ and GFP) were codon optimized ( Escherichia coli ), synthetized and cloned in pET22b plasmids by GeneArt (Waltham, MA, USA).

Techniques: Staining, Western Blot, Purification

Journal: Pharmaceutics

Article Title: In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics

doi: 10.3390/pharmaceutics12121217

Figure Lengend Snippet: Antimicrobial activity of assembled (PNPs) and unassembled soluble proteins (UPs) in vitro. ( A ) Effect on cell viability of increasing amounts of GWH1-GFP and GWH1-IFN-γ against E. coli and ( B ) S. aureus ( C ). Dynamic light scattering (DLS) analysis showing the nanoparticle disassembling of GWH1-GFP in presence of 0.2% N-lauroylsarcosine. ( D ) Comparative effect on cell viability of disassembled (promoted in presence of 0.2% N-lauroylsarcosine) and assembled GWH1-GFP against E. coli in vitro. IFN-γ and GFP were used as controls. Significant results are shown as * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001, multiple t test. All experiments were performed in duplicates. Bars represent the the mean ± standard deviation.

Article Snippet: All DNA sequences (GWH1-GFP, GWH1-IFN-γ, IFN-γ and GFP) were codon optimized ( Escherichia coli ), synthetized and cloned in pET22b plasmids by GeneArt (Waltham, MA, USA).

Techniques: Activity Assay, In Vitro, Standard Deviation

Journal: Pharmaceutics

Article Title: In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics

doi: 10.3390/pharmaceutics12121217

Figure Lengend Snippet: Ultrastructural characterization and analysis by electronic microscopy of purified inclusion bodies. Representative images of general fields and nanoparticle detail of entire IBs of GFP, GWH1-GFP, IFN-γ and GWH1-IFN-γ at two different magnifications, 11,000×/150,000× (FESEM) and 3000×/10,000× (TEM). Magnifications are equivalent in each micrograph to allow comparative visualization. The scale bars represent 500 nm.

Article Snippet: All DNA sequences (GWH1-GFP, GWH1-IFN-γ, IFN-γ and GFP) were codon optimized ( Escherichia coli ), synthetized and cloned in pET22b plasmids by GeneArt (Waltham, MA, USA).

Techniques: Microscopy, Purification

Journal: Pharmaceutics

Article Title: In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics

doi: 10.3390/pharmaceutics12121217

Figure Lengend Snippet: Antimicrobial effect of GFP and GWH1-GFP (PNPs) over S. aureus -infected mammary glands in vivo. Mammary glands were harvested and processed 8 h after the simultaneous administration of 100 CFU of S. aureus and 60 µmol/L of protein, respectively. Sodium bicarbonate buffer (166 mmol/L NaHCO 3 , 333 mmol/L NaCl) was used as control. The middle bars indicate median values for each group of glands (at least 6 glands) whereas the boxes specify quartiles Q1-Q3. The experiment was performed in triplicates. Significant results are shown as * p ≤ 0.05, ** p ≤ 0.01, one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test.

Article Snippet: All DNA sequences (GWH1-GFP, GWH1-IFN-γ, IFN-γ and GFP) were codon optimized ( Escherichia coli ), synthetized and cloned in pET22b plasmids by GeneArt (Waltham, MA, USA).

Techniques: Infection, In Vivo