Expression and purification of Clostridium perfringens beta-toxin glutathione S-transferase fusion protein

ABSTRACT Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.

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Glutathione S-Transferase/ras Fusion Protein: A Tool for Affinity Chromatography of ras Associated Proteins

ras Oncogenes ◽

10.1007/978-1-4757-1235-3_32 ◽

1989 ◽

pp. 255-260 ◽

Cited By ~ 1

Author(s):

Hiroshi Maruta

Keyword(s):

Fusion Protein ◽

Affinity Chromatography ◽

Protein A ◽

Glutathione S Transferase ◽

Associated Proteins

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Endothelial Binding of Beta Toxin to Small Intestinal Mucosal Endothelial Cells in Early Stages of Experimentally Induced Clostridium Perfringens Type C Enteritis in Pigs

Veterinary Pathology ◽

10.1177/0300985812461362 ◽

2012 ◽

Vol 50 (4) ◽

pp. 626-629 ◽

Cited By ~ 18

Author(s):

V. L. Schumacher ◽

A. Martel ◽

F. Pasmans ◽

F. Van Immerseel ◽

H. Posthaus

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Early Stages ◽

Type C ◽

Small Intestinal ◽

Beta Toxin ◽

Experimentally Induced

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Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger

Biochemistry and Cell Biology ◽

10.1139/o98-081 ◽

1998 ◽

Vol 76 (5) ◽

pp. 837-842 ◽

Cited By ~ 2

Author(s):

Daniel Gebreselassie ◽

Krishna Rajarathnam ◽

Larry Fliegel

Keyword(s):

Circular Dichroism ◽

Membrane Protein ◽

Fusion Protein ◽

Gel Electrophoresis ◽

Inclusion Bodies ◽

Cytoplasmic Domain ◽

Random Coil ◽

Glutathione S Transferase ◽

Carboxyl Terminal ◽

Circular Dichroïsm

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.Key words: Na+/H+ exchanger, pH regulation, membrane protein, circular dichroism.

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Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.93.17.9287 ◽

1996 ◽

Vol 93 (17) ◽

pp. 9287-9291 ◽

Cited By ~ 51

Author(s):

U. C. Vothknecht ◽

C. G. Kannangara ◽

D. von Wettstein

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Glutathione S Transferase ◽

Catalytically Active

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Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽

10.1182/blood.v84.4.1157.1157 ◽

1994 ◽

Vol 84 (4) ◽

pp. 1157-1163 ◽

Cited By ~ 13

Author(s):

EA Barron-Casella ◽

TS Kickler ◽

OC Rogers ◽

JF Casella

Keyword(s):

Amino Acids ◽

Fusion Protein ◽

Fusion Proteins ◽

Disulfide Bonds ◽

Affinity Purification ◽

Platelet Glycoprotein ◽

Enzyme Linked Immunosorbent Assay ◽

Glutathione S Transferase ◽

Amino Terminal ◽

Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

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