Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

2021 ◽  
Author(s):  
Cristina Hernandez Rollan ◽  
Kristoffer Bach Falkenberg ◽  
Maja Rennig ◽  
Andreas Birk Bertelsen ◽  
Morten Norholm
Keyword(s):  
Protein Production ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Strain Bl21 ◽  
Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

scholarly journals Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

1971 ◽  
Vol 123 (4) ◽  
pp. 501-505 ◽  
Author(s):  
J. W. Dale
Keyword(s):  
Escherichia Coli ◽  
Host Species ◽  
Host Bacterium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
R Factor ◽  
Relationship Of ◽  
The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

Synthesis, Characterization, and Antibacterial Evaluation of Curcumin-Sulfanilamide Compound

2020 ◽  
Vol 840 ◽  
pp. 265-269
Author(s):  
Nurjanah Nurjanah ◽  
Endang Saepudin
Keyword(s):  
Antibacterial Activity ◽  
Functional Group ◽  
Biological Activities ◽  
Curcuma Longa ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Ftir Spectrophotometry

Curcumin, a diarylheptanoids compound which isolated primary from Curcuma longa, exhibits a variety of exciting biological activities, including as an antibacterial agent. In the present study, a sulfanilamide-contained curcumin compound was synthesized and characterized to investigate the antibacterial activity against gram-positive bacteria S. aureus, B. subtilis and gram-negative bacteria E. coli. The characterization of the synthesized compound was determined by analysing peak absorbance, functional group, and molecular weight using mass spectroscopy, UV/Vis and FTIR spectrophotometry. Curcumin-sulfanilamide compound exhibited the best antibacterial activity against gram-negative bacteria compared to curcumin and the curcumin-derived compound containing isoxazole with inhibitory zone of 11 mm.

scholarly journals Discovery and characterization of a new family of lytic polysaccharide monooxygenases

2013 ◽  
Vol 10 (2) ◽  
pp. 122-126 ◽  
Author(s):  
Glyn R Hemsworth ◽  
Bernard Henrissat ◽  
Gideon J Davies ◽  
Paul H Walton
Keyword(s):  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

scholarly journals Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

2021 ◽  
Author(s):  
Valérie Biou ◽  
Ricardo Jorge D Adaixo ◽  
Mohamed Chami ◽  
Pierre-Damien Coureux ◽  
Benoist Laurent ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Molecular Motor ◽  
Opportunistic Pathogen ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Class ◽  
Heme Acquisition ◽  
Shed Light

ExbBD is part of a cytoplasmic membrane molecular motor driven by the proton-motive force. It belongs to the larger family of motors involved in nutriment import across the outer membrane of Gram-negative bacteria (ExbBD), flagellar rotation (MotAB) or late steps of cell division in Gram-negative bacteria (TolQR). ExbB and ExbD are integral membrane proteins with three (ExbB) or one (ExbD) transmembrane segment. Here we have solved by single-particle cryo-EM the structures of ExbB alone and of the ExbB-ExbD complex of the opportunistic pathogen Serratia marcescens. ExbBSm alone behaves as a stable pentamer, and the complex displays the ExbB5-ExbD2 stoichiometry. This is similar to what has been observed for ExbB-ExbD complexes from Escherichia coli and Pseudomonas savastanoi as well as MotAB complexes from various species. We identified residues located in the first TM of ExbBSm and ExbBEc that are likely involved in the interaction with TonB/HasB and that are essential for function. ExbBSm has a ca. 40 residues long periplasmic extension absent in E. coli. Such long ExbBs are found in some Gammaproteobacteria, and several genera of Alphaproteobacteria. We show that this extension interacts with HasB, a dedicated TonB paralog from the heme acquisition system (Has) from S. marcescens. We also show that it is involved in heme acquisition via the Has system from S. marcescens. ExbBSm represents thus a new class of ExbB protein and our results shed light on the specificity determinants between the ExbB-ExbD complex and their associated TonB partners.

scholarly journals Directed evolution for improved total secretory protein production in Escherichia coli

2020 ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Protein Production ◽  
Secretory Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Gram Negative Bacteria ◽  
Carrier Proteins ◽  
Gram Negative ◽  
E Coli

ABSTRACTProduction of secretory protein in Gram-negative bacteria simplifies downstream processing in recombinant protein production, accelerates protein engineering, and advances synthetic biology. Signal peptides and secretory carrier proteins are commonly used to effect the secretion of heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have successfully applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate the directed evolution workflow. In this study, we applied directed evolution on OsmY to enhance its total secretory protein production.After just one round of directed evolution followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C). Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype.This is the first report of applying directed evolution on a carrier protein to enhance total secretory protein production. The methodology can be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli and other bacteria. In this study, OsmY(M3) improved the production of three proteins, originating from diverse organisms and with diverse properties, in secreted form, clearly demonstrating its wide-ranging applications.

scholarly journals Evaluation the Biological Activity of Nano- Gentamicin Prepared and Characterization of Nanoparticles

2021 ◽  
Vol 8 (2) ◽  
pp. 84-90
Author(s):  
Rana Kamal ◽  
Quraish A. Kahdhum ◽  
Awatif I. Mohammed ◽  
Ahmed J. Essa ◽  
Maan A. Abd Elhamid ◽  
...  
Keyword(s):  
Biological Activity ◽  
Antibacterial Effect ◽  
Sol Gel ◽  
Homogeneous Distribution ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Average Size

Gentamycin is a broad-spectrum antibacterial against Gram-positive and Gram-negative bacteria. Gentamycin Sulfate is a white powder that is freely soluble in water and insoluble in alcohol, acetone, chloroform and ether. The study aims to determine the characterization and the biological activity of gentamicin in the Nano-form prepared by Sol-gel application using an ultrasound device by aqueous solution at temperature of 80°. Nano gentamycin examined by Scanning electron microscopy (SEM), transmission (TEM) and the antibacterial effect test of both Nano and stander gentamycin were analyzed. The results showing that the average size of gentamycin nanoparticles is 68.51 nanometers with homogeneous distribution. The antibacterial effect of Nano gentamycin take a wide inhibition range of Gram-positive and Gram-negative bacteria compared to the standard substance, the inhibition diameters are for E. coli (29 mm) and for St. epidermidis (27 mm). In the other side the inhibitory ability for standard gentamicin for St. epidrmidis and E. coli were 18 mm and 20 mm, respectively.

scholarly journals PREVALENCE AND MOLECULAR CHARACTERIZATION OF blaCTX-M-15-PRODUCING PATHOGENIC GRAM-NEGATIVE BACTERIA FROM VARIOUS CLINICAL SAMPLES

2018 ◽  
Vol 11 (10) ◽  
pp. 386
Author(s):  
Sindhuja S ◽  
Sureshkumar Bt ◽  
Janaki S ◽  
Thenmozhi S
Keyword(s):  
Molecular Characterization ◽  
Pathogenic Bacteria ◽  
Disk Diffusion ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli

Objective: The objective of this study was to describe the prevalence and molecular characterization of blaCTX-M-15-producing pathogenic Gram-negative bacteria from various clinical samples isolated from clinically suspected patients.Methods: In this study, clinical samples of urine, stool, sputum, and pus were collected from 244 patients with nosocomial infections. The phenotypic identification of extended-spectrum β-lactamases (ESBL) was confirmed by double-disk synergy test and combined disk diffusion test. In vitro, the susceptibility pattern of antimicrobial agents against pathogenic isolates was performed by Kirby–Bauer disk diffusion method. The identification of blaCTX-M-15-producing Escherichia coli was assessed by polymerase chain reaction method.Results: The frequency of ESBL-producing pathogenic bacteria from screened was 6 (46.15%). In vitro, susceptibility to pathogenic bacteria showed that the majority of isolates were highly susceptible to amoxicillin-clavulanic acid (97.87%), ofloxacin (93.33%), and Pseudomonas aeruginosa showed 100% sensitive to ceftazidime, cefotaxime, cefixime, cefoperazone, and meropenem (92.30%). The rates of resistance to other antibiotics varied from <26.66%. Among six tested isolates, only one E. coli isolates showed blaCTX-M-15 gene.Conclusion: Due to the increase of E. coli with multiple ESBL genes, continuous surveillance should be needed in clinical field to use of appropriate antibiotics and the control of infections.

scholarly journals Characterization of lptA and lptB, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 244-253 ◽  
Author(s):  
Paola Sperandeo ◽  
Rachele Cescutti ◽  
Riccardo Villa ◽  
Cristiano Di Benedetto ◽  
Daniela Candia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
De Novo ◽  
Essential Genes ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
E Coli ◽  
Σ Factor

ABSTRACT The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

Disinfection of Fruits with Activated Water

2019 ◽  
Vol 10 ◽  
pp. 1864-1872
Author(s):  
Prof. Teodora P. Popova
Keyword(s):  
Antimicrobial Activity ◽  
Aqueous Solutions ◽  
Antimicrobial Properties ◽  
The Other ◽  
Gram Negative Bacteria ◽  
High Antimicrobial Activity ◽  
Gram Negative ◽  
E Coli ◽  
Number Of Microorganisms ◽  
Lower Effect

The effect of ionized aqueous solutions (anolytes and catholyte) in the processing of fruits (cherries, morellos, and strawberries) for decontamination has been tested. Freshly prepared analytes and catholyte without the addition of salts were used, as well as stored for 7 months anolytes, prepared with 0.5% NaCl and a combination of 0.5% NaCl and 0.5% Na2CO3. The anolyte prepared with a combination of 0.5% NaCl and 0.5% Na2CO3, as well as the anolyte obtained with 0.5% NaCl, exhibit high antimicrobial activity against the surface microflora of strawberries, cherries, and sour cherries. They inactivate E. coli for 15 minutes. The other species of the fam. Enterobacteriaceae were also affected to the maximum extent, as is the total number of microorganisms, especially in cherries and sour cherries. Even stored for 7 months, they largely retain their antimicrobial properties. Anolyte and catholyte, obtained without the addition of salts, showed a lower effect on the total number of microorganisms, but had a significant effect on Gram-negative bacteria, and especially with regard to the sanitary indicative E. coli.

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