scholarly journals Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Fatemah Bakhshi ◽  
Reza Pilehchian Langroudi ◽  
Bahram Golestani Imani
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Clostridium Perfringens ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Toxin Gene ◽  
Type B ◽  
Iptg Induction ◽  
E Coli ◽  
Beta Toxin

Clostridium perfringens beta toxin is only produced by types B and C and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. We compared the expression of C. perfringens type B vaccine strain recombinant beta toxin gene in the Escherichia coli strains RosettaTM(DE3) and BL21(DE3). The beta toxin gene was extracted from pJETβ and ligated with pET22b(+). pET22β was transformed into E. coli strains BL21(DE3) and RosettaTM(DE3). Recombinant protein was expressed as a soluble protein after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in strain RosettaTM(DE3) but not in BL21(DE3). Expression was optimised by growing recombinant cells at 37 °C and at an induction of 0.5 mM, 1 mM, 1.5 mM IPTG. Expression was evaluated using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified via Ni-NTA and was analysed using western blot. We concluded that E. coli strain RosettaTM(DE3) can enhance the expression of C. perfringens recombinant beta toxin.Keywords: C. perfringens beta toxin (CPB); expression; RosettaTM; BL21

scholarly journals MOLECULAR EXPRESSION OF WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY MEMBER 4 GENE USING Escherichia coli BL21

2017 ◽  
Vol 11 (1) ◽  
pp. 11-14
Author(s):  
Agung Janika Sitasiwi ◽  
Wayan Tunas Artama ◽  
Agung Budiyanto ◽  
Edy Dharmana
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Dna ◽  
Integration Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Negative Control ◽  
Luria Bertani ◽  
Iptg Induction ◽  
E Coli

This research was conducted to find out the Wnt4 recombinant proteins which expressed by Escherichia coli (E. coli) BL21 carrying the recombinant DNA wnt4 (E. coli transformation). Research materials were E. coli BL21 transformation and E. coli BL21 non-transformation (negative control). The expression of recombinant protein was conducted by culturing E. coli for 24 hours in Luria-Bertani (LB) media with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Recombinant protein was isolated by sonication of pellet bacteria. Protein analysis performed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that recombinant protein with a molecular weight of 33 kDa has been expressed by E. coli BL21 transformation successfully. 

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

scholarly journals Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

2006 ◽  
Vol 74 (4) ◽  
pp. 2233-2244 ◽  
Author(s):  
Alison S. Low ◽  
Francis Dziva ◽  
Alfredo G. Torres ◽  
Jessenya L. Martinez ◽  
Tracy Rosser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
Induced Expression ◽  
E Coli ◽  
Extracellular Matrix Components ◽  
K 12

ABSTRACT Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

scholarly journals Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid

2008 ◽  
Vol 190 (21) ◽  
pp. 7178-7188 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Jihong Li ◽  
Sameera Sayeed ◽  
Shigeru Akimoto ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Open Reading Frames ◽  
Toxin Gene ◽  
Type B ◽  
Clostridium Perfringens Enterotoxin ◽  
Type D ◽  
Transfer Genes ◽  
Epsilon Toxin ◽  
Beta2 Toxin ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

scholarly journals Cloning and expression most expected antigenic fragment of beta-toxin gene from Clostridium perfringens type B

2016 ◽  
Vol 05 (05) ◽  
pp. 491-494
Author(s):  
Maryam Gholami ◽  
Mohsen Fathi Najafi ◽  
Mohammad Rabbani Khorasgani ◽  
Behjat Majidi
Keyword(s):  
Clostridium Perfringens ◽  
Cloning And Expression ◽  
Toxin Gene ◽  
Type B ◽  
Beta Toxin

scholarly journals Media Optimization for Production of Recombinant Carrier Protein (CRM197) in Escherichia coli

2021 ◽  
Vol 13 (1) ◽  
pp. 283-297
Author(s):  
S. Shukla ◽  
D. Mishra
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Luria Bertani ◽  
Lag Phase ◽  
Carrier Protein ◽  
Scale Production ◽  
E Coli ◽  
Defined Media

Since the advent of vaccines, the mankind has benefited from the same and has been able to curb the mortality rate around the globe. Amongst different types of available vaccines, polysaccharide based vaccines are very widely used against various infectious diseases. The polysaccharide vaccines need to be conjugated with a carrier protein to make the vaccine more immunogenic. Recombinant Escherichia coli cells are the organism of choice for large scale production of a carrier protein because of its widely studied scientific aspects. In the present study, for proof of concept, the recombinant E. coli cells were cultured in Luria-Bertani media to check the expression of rCRM197. At 80L scale, it was observed that when recombinant E. coli cells were grown in a chemically defined media, it resulted in inconsistent growth and a long lag phase. When the defined media was supplemented with yeast extract, the lag phase of the culture was substantially reduced and the maximum growth of the culture was achieved. Protein expression was checked using SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) and Western blot technique. The optimized media resulted in a robust fermentation process to achieve high cell density and maximum biomass for the production of recombinant protein.

Isolation and Purification of Colicin ECL 12, a Bacteriocin That Inhibits Strains of Escherichia coli O157:H7†

1997 ◽  
Vol 60 (12) ◽  
pp. 1520-1528 ◽  
Author(s):  
WANDA J. LYON ◽  
DENNIS G. OLSON
Keyword(s):  
Escherichia Coli ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Viable Cell ◽  
Exchange Chromatography ◽  
Isolation And Purification ◽  
E Coli ◽  
Log Reduction ◽  
Cell Counts

A swine fecal isolate, identified as Escherichia coli ECL12, was found to produce an antimicrobial substance designated as colicin ECL12. Colicin ECL12 was inhibitory against 20 strains of E. coli O157:H7 previously isolated from both human and bovine feces. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Colicin ECL12 was sensitive to several proteolytic enzymes. Adsorption of colicin ECL12 to sensitive cells of E. coli O157:H7 was bactericidal, resulting in a 2 log reduction in viable cell counts. Colicin ECL12 was purified from strain ECL12 by cell extraction and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of colicin ECL12 resolved a single protein with a molecular weight of approximately 65,000.

scholarly journals COMPARISON OF EXTRACELLULAR SECRETION OF RECOMBINANT HUMAN EPIDERMAL GROWTH FACTOR USING TORA AND PELB SIGNAL PEPTIDES IN ESCHERICHIA COLI BL21 (DE3)

2019 ◽  
pp. 81-84 ◽  
Author(s):  
RIMA MELATI ◽  
ANNISA INDRIYANI ◽  
SHABARNI GAFFAR ◽  
SRIWIDODO ◽  
IMAN PERMANA MAKSUM
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Signal Peptides ◽  
Extracellular Expression ◽  
E Coli ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth

Objective: The objective of this study was to evaluate two signal peptides (TorA and PelB), representing the most common secretion pathways in Escherichia coli, for their ability to secrete recombinant human epidermal growth factor (rhEGF) protein in the extracellular expression. Methods: E. coli BL21 (DE3) as the host cell to be transformed using recombinant plasmid pD881-TorA the consensus already containing hEGF gene and the signal peptide TorA or PelB, then expressed by L-rhamnose induction. rhEGF purified by heat treatment and ion-exchange chromatography. The hEGF protein was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ELISA. Results: The result showed that PelB was secreting more hEGF protein compared to TorA with protein expression results of 48.2 μg/L and purification results of 0.360 μg/L, with a purity level of 83%. Conclusion: The results of this study explain in extracellular expression of hEGF protein in E. coli, PelB helps hEGF protein secretion to culture media better than TorA.

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