Construction and Preservation of a Stable and Highly Expressed Recombinant Helicobacter pylori Vacuolating Cytotoxin A with Apoptotic Activity

Abstract Background: VacA is the only exocrine toxin of H. pylori, which plays a very important role in the customization of H. pylori in the gastric mucosa, contributing to the pathogenesis of gastritis-cancer. Studies about vacA structure and function was hindered by the lack of efficient production system. In this study, we developed methodology for expression, purification and stable storage of a functionally active vacA toxin in E.coli.Results: A 2502-bp fragment and vacA gene were identified. An 89.7-kDa VacA34-854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.Conclusions: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

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Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity

BMC Microbiology ◽

10.1186/s12866-021-02262-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ling-Zhi Yuan ◽

Xiao Shi ◽

Dan Tang ◽

Shao-Peng Zheng ◽

Zhi-Ming Xiao ◽

...

Keyword(s):

Acetic Acid ◽

Recombinant Protein ◽

Biologically Active ◽

Acid Buffer ◽

Inverted Microscope ◽

Acetic Acid Buffer ◽

Apoptotic Activity ◽

H Pylori ◽

Induced Apoptosis

Abstract Background H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer. Results A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA34–854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay. Conclusions A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

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pH Measurement Problems Affecting Assay of Carcinoembryonic Antigen

Clinical Chemistry ◽

10.1093/clinchem/21.2.255 ◽

1975 ◽

Vol 21 (2) ◽

pp. 255-257 ◽

Cited By ~ 4

Author(s):

Jack H Ladenson ◽

C Elliott Bell

Keyword(s):

Acetic Acid ◽

Carcinoembryonic Antigen ◽

Ammonium Acetate ◽

Electrode System ◽

Acid Buffer ◽

Ph Values ◽

Blood Ph ◽

Acetic Acid Buffer ◽

Ph Electrodes ◽

Commercial Kit

Abstract Measurement with combination pH electrodes of the pH of the dilute buffers used in a commercial kit (CEA-Roche) for assay of carcinoembryonic antigen resulted in pH values 0.1 to 0.3 unit lower than pH values measured on an electrode system with a capillary junction. If the pH values of these buffers were adjusted, based on such measurements, an error in the assay of 0.2 to 0.6 ng/ml in the 1.5-3.0 ng/ml range would result. We recommend that the pH of dialyzed samples and of the working ethylenediaminetetraacetate and ammonium acetate-acetic acid buffers be monitored with pH electrodes that have a capillary junction between sample and saturated KCl, as is true of most blood-pH instruments. We also recommend use of a 1 mol/liter rather than 2.5 mol/liter stock ammonium acetate-acetic acid buffer, because of the closer similarity of the pH of buffers at this molarity to those at 0.01 mol/liter.

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Apoptotic Signaling Pathway Activated byHelicobacter pylori Infection and Increase of Apoptosis-Inducing Activity under Serum-Starved Conditions

Infection and Immunity ◽

10.1128/iai.69.5.3181-3189.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3181-3189 ◽

Cited By ~ 37

Author(s):

Keigo Shibayama ◽

Yohei Doi ◽

Naohiro Shibata ◽

Tetsuya Yagi ◽

Toshi Nada ◽

...

Keyword(s):

Membrane Fraction ◽

Proteinase K ◽

Caspase 8 ◽

Peptic Ulcers ◽

Caspase 9 ◽

Effector Caspase ◽

Epithelial Cell Apoptosis ◽

Apoptotic Activity ◽

H Pylori ◽

Induced Apoptosis

ABSTRACT The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pyloriinfection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressedH. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.

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