One-step Negative Chromatographic Purification of <em>Helicobacter pylori</em> Neutrophil-activating Protein Overexpressed in <em>Escherichia coli</em> in Batch Mode

Abstract

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established. In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin. This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

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

“…Recently, we have developed a one-step diethylaminoethyl (DEAE) negative mode chromatography to purify native recombinant HP-NAP overexpressed in Escherichia coli ( E . coli ) with high purity and high yield by collecting the unbound fraction at pH 8.0 [ 23 , 24 ]. During the purification at 4°C, a minimal amount of HP-NAP was detected in the elution fraction at pH 8.0, whereas a substantial amount of HP-NAP was present in the elution at pH 7.0 and 9.0 [ 23 ].…”