Journal: Scientific Reports

Article Title: FimH-based display of functional eukaryotic proteins on bacteria surfaces

doi: 10.1038/s41598-019-44883-z

Figure Lengend Snippet: Design and expression of POI-FimH fusion proteins. ( A ) The Gaussia Luciferase (GLuc) is N-linked to the FimH protein and thereby becomes part of the fimbriae on the bacterial surface. By use of the bacterial pet11d vector, the expression of the GLuc-FimH fusion protein is induced by incubation with IPTG. ( B ) Schematic representation of the expression cassettes for the FimH fusion proteins with human EGF, human TGF-α and human epiregulin EPRG, respectively. Gly, Glycine-(Gly-Gly-Gly-Gly-Ser) 3 -linker; cMyc, c-Myc tag; CS, HRV3C protease cleavage site. ( C ) The expression of the POI-FimH protein on the surface of E. coli was induced by incubation with IPTG for 2 hrs and recorded by flow cytometry. Bacteria containing the GLuc-FimH expression vector were stained with a Gaussia luciferase-specific rabbit antibody and detected with a PE-coupled anti-rabbit antibody (bold histogram). An isotype-matched PE-conjugated antibody (light histogram) and non-modified E. coli (wt) served as controls. The expression of EGF-FimH, TGFα-FimH and Epiregulin-FimH on the surface of engineered E. coli bacteria was induced by IPTG. The bacteria were stained with a PE labeled c-Myc-specific mouse antibody and the proteins were recorded by flow cytometry (bold histograms). An isotype-matched PE-conjugated antibody (light histograms) served as control. All experiments were performed in triplicates and a representative histogram is shown.

Article Snippet: The detection of the proteins of interest was performed by using ELISA kits specific for human EGF, TGF-α and EPRG, respectively (CUSABIO BIOTECH, Houston, TX, USA).

Techniques: Expressing, Luciferase, Plasmid Preparation, Incubation, Ubiquitin Proteomics, Flow Cytometry, Bacteria, Staining, Modification, Labeling, Control

Journal: Scientific Reports

Article Title: FimH-based display of functional eukaryotic proteins on bacteria surfaces

doi: 10.1038/s41598-019-44883-z

Figure Lengend Snippet: Recombinant EGFR ligands produced by transformed E. coli induce EGFR receptor phosphorylation. ( A ) E. coli bacteria were engineered with FimH fusion proteins linked with the EGFR ligand epiregulin, EGF or TGF-α, respectively. Displayed EGFR ligands were proteolytically released from the bacteria surface and detected by ELISA. The analysis was performed in triplicates for each condition and the entire experiment repeated at least twice; representative data are shown. ( B ) Proteolytically released proteins of interest, i.e., EGF, TGF-α and epiregulin were tested for binding to the EGF receptor (EGFR). Therefore, microtiter wells were coated with the bacteria produced EGF, TGF-α and epiregulin, respectively, and incubated with the human EGFR-hIgG (filled symbols) and mouse EGFR-IgG (open symbols) fusion protein to test for specific binding. A human IgG fusion protein of irrelevant specificity (bold line) served as control. Bound EGFR-IgG fusion protein was detected by a biotin-labeled human IgG-specific antibody. ( C ) EGFR + 293T cells were incubated in the presence of proteolytically released EGF, TGF-α or EPRG, respectively, and tested for EGFR (Tyr1173) phosphorylation by flow cytometry. The mean fluorescence intensity (MFI) after incubation with the EGFR ligands is shown. All experiments were performed in triplicates for each condition and repeated at least twice; a representative experiment is shown.

Article Snippet: The detection of the proteins of interest was performed by using ELISA kits specific for human EGF, TGF-α and EPRG, respectively (CUSABIO BIOTECH, Houston, TX, USA).

Techniques: Recombinant, Produced, Transformation Assay, Phospho-proteomics, Bacteria, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Control, Labeling, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

doi: 10.1371/journal.pone.0160933

Figure Lengend Snippet: Differential effects of SMLEF bipolar pulses, SMHEF pulses and thrombin on PRP activation, growth factor release, and cell proliferation.

Article Snippet: Growth factor-dependent cell proliferation was confirmed by addition of purified recombinant human EGF (Lonza, Portsmouth, NH, USA).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

doi: 10.1371/journal.pone.0160933

Figure Lengend Snippet: PRP, treated as described in the Methods, was centrifuged, the supernatant recovered and assayed for pro- and anti-angiogenic factors by ELISA and for stimulation of cell proliferation using serum-starved epithelial cells (MCF10A). Cell proliferation in response to the plasma supernatants of unactivated or activated PRP (panel E) is normalized to that obtained with supernatants of unactivated PRP. Purified recombinant human EGF 100 ng/mL added to serum-free media increased cell proliferation 1.75-fold relative to media alone (data not shown). Results shown are means ± SEM, n = 5. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Growth factor-dependent cell proliferation was confirmed by addition of purified recombinant human EGF (Lonza, Portsmouth, NH, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Purification, Recombinant