Journal: PLoS ONE

Article Title: Identification, Expression and Activity Analyses of Five Novel Duck Beta-Defensins

doi: 10.1371/journal.pone.0047743

Figure Lengend Snippet: SDS–PAGE analysis of glutathione-S-transferase (GST)-tagged recombinant Apl_avian β-defensin (Apl_AvBD) proteins expressed in E. coli BL21 (DE3) cells. (A) total protein from BL21 containing Apl_AvBD1, 3, 5, 6, 16, and GST with IPTG induction, respectively; (B) inclusion bodies with Apl_AvBD1, 3, 5, 6, 16, and GST respectively; (C) purified proteins of Apl_AvBD1, 3, 5, 6, 16, and GST with IPTG induction, respectively. IPTG, isopropyl-beta-D-thiogalactoside.

Article Snippet: Antibacterial Activity Colony counting assays were performed according to the methods used in previous studies to investigate the antimicrobial activities of glutathione-S-transferase (GST) and the recombinant AvBDs (rAvBDs), and synthetic AvBDs (sAvBDs) from ducks against four strains of Gram-positive bacteria: Micrococcus tetragenus (ATCC2835), Lactobacillus (ATCC33222), Staphylococcus aureus (ATCC 29213), and Bacillus cereus (ATCC 9193); and eight strains of Gram-negative bacteria: Bordetella bronchiseptica (S80103), Proteus mirabilis (ATCC29245), Pseudomonas aeruginosa (ATCC 9027), Pasteurella multocida (ATCC 6529), E. coli BL21 (DE3), Salmonella pullorum (C79-11-S11), Salmonella choleraesuis (CVCC 2140), and Salmonella enteritidis (ATCC 3021).

Techniques: SDS Page, Recombinant, Purification

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top-Down LESA Mass Spectrometry Protein Analysis of Gram-Positive and Gram-Negative Bacteria

doi: 10.1007/s13361-017-1718-8

Figure Lengend Snippet: Liquid extraction surface analysis (LESA) mass spectra of seven bacterial strains: Escherichia coli K-12, Escherichia coli BL21 mCherry, Pseudomonas aeruginosa PS1054, Staphylococcus aureus MSSA476, Streptococcus pneumoniae D39, Streptococcus oralis ATCC 35037, and Streptococcus gordonii ATCC 35105. All mass spectra were acquired immediately following incubation of the colonies at 37 °C, with use of the 40:60:1 acetonitrile–water–formic acid solvent system for Gram-negative bacteria and the 50:45:5 variant for Gram-positive bacteria. The streptococci were incubated under semianaerobic conditions for optimum growth; the remaining strains were grown in open air

Article Snippet: General Overview of the Mass Spectra Figure shows representative mass spectra of seven bacterial colonies corresponding to seven strains: Gram-negative E. coli K-12, E. coli BL21 mCherry, and P. aeruginosa PS1054, and Gram-positive S. aureus MSSA476, S. pneumoniae D39, S. oralis ATCC 35037, and S. gordonii ATCC 35105.

Techniques: Incubation, Variant Assay

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor

doi: 10.1016/j.jgeb.2016.12.006

Figure Lengend Snippet: Comparison of expression levels of TNFR ED, EPO and SK in different hosts at 37 °C on induction with 1 mM IPTG for E. coli BL21 (DE3) and BL21 (DE3) pLys S strains and 0.3 M NaCl for E. coli GJ1158 strains at (A) 0.6 OD 600 , (B) 1.0 OD. Basal level expression was given a minimal value of 0.5%.

Article Snippet: E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S (Novagen,) are IPTG inducible strains in which the T7 RNA Polymerase gene is under the control of a Lac UV5 promoter.

Techniques: Expressing

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor

doi: 10.1016/j.jgeb.2016.12.006

Figure Lengend Snippet: Immunoblot analysis of total protein of induced E. coli BL21 (DE3) cellsharboring the recombinant plasmids using anti-His antibodies . Lanes 1 – Protein marker, 2 – pRSET A vector control induced, 3 – pRSET-TNFR ED uninduced, 4 – pRSET-TNFR ED induced, 5 – pRSET EPO-uninduced, 6 – pRSET EPO-induced, 7 – pRSET SK uninduced, 8 – pRSET SK induced.

Article Snippet: E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S (Novagen,) are IPTG inducible strains in which the T7 RNA Polymerase gene is under the control of a Lac UV5 promoter.

Techniques: Recombinant, Marker, Plasmid Preparation

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