Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein.

Zhi-Wei Hong,Timothy Pan,Hua-Wen Fu,Huey-Fen Tzeng,Yu-Chi Yang

doi:10.1371/journal.pone.0173632

Zhi-Wei Hong, Timothy Pan + Show 3 more

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https://doi.org/10.1371/journal.pone.0173632

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Abstract

Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification.

Highlights

Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic pathogen, which colonizes the gastric mucosa of human stomach
The recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) expressed in E. coli was subjected to batch chromatography using DEAE Sepharose and DEAE Sephadex resins under these conditions
The UV absorbance was recorded at 220 nm. 4-Methoxybenzyl alcohol (4mBA) was used as the neutral electroosmotic flow (EOF) marker, which migrated as a peak at μe = 0 for all electropherograms

Summary

Introduction

Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic pathogen, which colonizes the gastric mucosa of human stomach. HP-NAP was first identified in the water extract of H. pylori for its ability to promote neutrophil adhesion to endothelial cells and production of reactive oxygen species (ROS) by neutrophils [5] This protein is capable of activating neutrophils, monocytes and mast cells to secrete pro-inflammatory cytokines and inflammatory mediators [6,7,8,9,10], which could further activate gastric inflammation and result in the damage of gastric mucosa. High amounts of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were found to be produced by antigen-specific gastric Th cells upon HP-NAP stimulation [7]. Both innate and adaptive immune responses induced by HP-NAP contribute to the pathological outcome during H. pylori infection

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