Helicobacter pylori is closely associated with chronic gastritis, peptic ulcer disease, and gastric adenocarcinoma. Helicobacter pylori neutrophil-activating protein (HP-NAP), a virulence factor of Helicobacter pylori, plays an important role in pathogenesis of Helicobacter pylori infection. Since HP-NAP has been proposed as a candidate vaccine against Helicobacter pylori infection, an efficient way to obtain pure HP-NAP needs to be developed. In the present invention, recombinant HP-NAP expressed in Bacillus subtilis and Escherichia coli was purified through a single step of DEAE SEPHADEX ion-exchange chromatography with high purity. Also, purified recombinant HP-NAP was able to stimulate neutrophils to produce reactive oxygen species. Thus, recombinant HP-NAP obtained from our Bacillus subtilis expression system and Escherichia coli expression system is functionally active. Furthermore, this one-step negative purification method should provide an efficient way to purify recombinant HP-NAP expressed in Bacillus subtilis and Escherichia coli for basic studies, vaccine development, or drug design.