scholarly journals Cloning, expressing and purification of recombinant C-terminal PMT of Pasteurella multocida

2019 ◽  
Vol 18 (5) ◽  
pp. 62-69
Author(s):  
Hung K. Vu
Keyword(s):  
Escherichia Coli ◽  
Pasteurella Multocida ◽  
Protein Band ◽  
Recombinant Vaccine ◽  
Insoluble Protein ◽  
Terminal Protein ◽  
Promising Candidate ◽  
Heat Stable ◽  
Sds Page ◽  
Pasteurella Multocida Toxin

Pasteurella multocida strains produce heat-stable toxin Pasteurella Multocida Toxin (PMT) which is the main virulence factor causing pasteurellosis in pigs. Therefore, PMT protein is considered as a promising candidate to study recombinant vaccine against pasteurellosis in pigs. In this study, we cloned the nucleotide sequence coding C-terminal fragment of PMT (tPMT-780), from acid amine 506 to acid amine 1285, into pRSET-A vector and expressed it on Escherichia coli BL21 bacteria. The SDS-PAGE result showed that tPMT-C780 was well-expressed by IPTG 1mM induction and determined to be insoluble protein, therefore it was purified by denaturation method using Ni-NTA beads. The protein elution samples contained only one protein band similar with the size of tPMT-C780 and positively reacted with PMT-specific antibody. These results suggested that we were successful in expressing and purifying C-terminal protein of PMT toxin, tPMT-C780. This is the primary material for further studies to produce recombinant vaccine against pasteurellosis in pigs.

scholarly journals Physiochemical properties of a heat-stable enterotoxin produced by Escherichia coli of human origin

1980 ◽  
Vol 28 (3) ◽  
pp. 1051-1053
Author(s):  
G L Madsen ◽  
F C Knoop
Keyword(s):  
Escherichia Coli ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Protein Band ◽  
Biologically Active ◽  
Enterotoxigenic Escherichia Coli ◽  
Physiochemical Properties ◽  
Heat Stable ◽  
Single Protein

Concentrated cell-free filtrates, prepared from a human strain of enterotoxigenic Escherichia coli, were subjected to isoelectric focusing, molecular sieve chromatography, and polyacrylamide disc gel electrophoresis. Isoelectric focusing in a pH 3 to 5 gradient resulted in two biologically active peaks, I and II, that electrofocused at pI 1.5 and 3.8, respectively. Molecular sieve chromatography of the major enterotoxic peak (II) at pH 3.8 indicated a molecular weight of 2,500. Polyacrylamide disc gel electrophoresis of ST revealed a single protein band containing enterotoxic activity.

scholarly journals Purification and Characterization of Glutathione Binding Protein GsiB from Escherichia coli

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Zhongshan Wang ◽  
Xiaokun Xia ◽  
Meixian Zhang ◽  
Jiawei Fang ◽  
Yanqiang Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Knockout ◽  
Binding Protein ◽  
Gel Filtration ◽  
Direct Interaction ◽  
Protein Band ◽  
Sulfur Source ◽  
E Coli ◽  
Sds Page ◽  
Glutathione Binding

Objectives. To purify and characterize the glutathione binding protein GsiB of glutathione importer (GSI) in Escherichia coli (E. coli). Results. The coding sequence of GsiB was cloned from E. coli MG1655 and expressed in BL21(DE3). GsiB protein was expressed and purified to homogeneity using Ni-affinity and gel filtration chromatography. SDS-PAGE of purified GsiB showed a single protein band of molecular mass 56 kDa, while native gel showed two bands around 56 kDa and 110 kDa. Gene knockout showed that GsiB was essential for GSI mediated glutathione import. Interactions of GsiA, B, C, and D were determined using bacterial two-hybrid method. Without glutathione, GsiB showed no direct interaction with the other three proteins. However, GsiB could interact with GsiC and GsiD when using glutathione as sole sulfur source. Conclusions. GsiB functions in E. coli was characterized which could help elucidate the glutathione import mechanism in gram-negative bacteria.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

Comparison of colony and stool blots for detection of enteropathogens by DNA probes

1991 ◽  
Vol 37 (5) ◽  
pp. 407-410
Author(s):  
Mônica A. M. Vieira ◽  
Beatriz E. C. Guth ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Sensitivity And Specificity ◽  
Dna Probes ◽  
Type I ◽  
Enteropathogenic Escherichia Coli ◽  
Key Words Dna ◽  
E Coli ◽  
Heat Stable

DNA probes that identify genes coding for heat-labile type I (LT-I) and heat-stable type 1 (ST-I) enterotoxins, enteropathogenic Escherichia coli adherence factor (EAF), and Shigella-like, invasiveness (INV) are used to evaluate the sensitivity and specificity of stool blots in comparison with the sensitivity and specificity of colony blots in detecting enteropathoghens. The sensitivities of the probes in stool blots are 91.7% for the LT-I probe, 76.9% for the ST-I probes, 78.9% for the EAF probe, and 45.5% for the INV probe. The specificity of all probes is higher than 95%. In general, the stool blot method identifies as many if not more LT-I-, ST-I-, and EAF-producing E. coli infections than the colony blots. Key words: DNA probes, stool blots, enteropathogens, diagnosis.

Use of a Multiplex PCR System for the Simultaneous Detection of Heat Labile Toxin I and Heat Stable Toxin II Genes of Enterotoxigenic Escherichia coli in Skim Milk and Porcine Stool

1998 ◽  
Vol 61 (2) ◽  
pp. 141-145 ◽  
Author(s):  
HAU-YANG TSEN ◽  
LIANG-ZHAO JIAN ◽  
WAN-RONG CHI
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Skim Milk ◽  
Pcr Primers ◽  
Enterotoxigenic Escherichia Coli ◽  
Farm Animals ◽  
Target Cells ◽  
Heat Stable ◽  
Heat Labile

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT I- and ST II-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 100 cells per ml of the sample could be detected. Without the preculture step, 104 CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR System can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

Isolation and characterization of the heat stable enterotoxin from a pathogenic bovine strain of Escherichia coli

1984 ◽  
Vol 9 (4) ◽  
pp. 399-414 ◽  
Author(s):  
Charles Gerday ◽  
Marianne Herman ◽  
Jacques Olivy ◽  
Nicole Gerardin-Otthiers ◽  
Dominique Art ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Stable ◽  
Isolation And Characterization ◽  
Bovine Strain
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