Abstract
Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in inflammatory response of gastric mucosal diseases induced by Helicobacter pylori. Recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis) can be purified by using one-step DEAE anion-exchange chromatography by collecting the unbound fraction at pH 8.0. To understand why HP-NAP did not bind DEAE resins during the ion-exchange purification process at the pH above its isoelectric point, HP-NAP expressed in Escherichia coli (E. coli) was subjected to DEAE anion-exchange purification at the pH range from 7.0 to 9.0, which are exceeded the pI of HP-NAP, to examine if HP-NAP is present in the elution fraction. The recombinant HP-NAP was mainly present in the unbound fraction at pH 7.5 to 9.0 but not at pH 7.0. At pH 8.0, most of the recombinant HP-NAP was present in the unbound fraction and little amount of recombinant HP-NAP was detected in the elution fraction. Under the optimal condition at pH 8.0, recombinant HP-NAP from E. coli can be obtained with a purity of 95.97% by using this one-step negative purification with DEAE Sephadex anion-exchange resins. The purified recombinant HP-NAP was characterized as an oligomeric protein with a secondary structure of -helix and was able to trigger ROS production from neutrophils. To clarify whether HP-NAP interacts with DEAE resins at pH 7.0 to 9.0, HP-NAP obtained from the unbound fraction was incubated with DEAE resins to examine if HP-NAP could bind DEAE resins. However, the purified HP-NAP was present in the unbound fraction at pH 7.0 to 9.0 in the absence of impure proteins. It is assumed that HP-NAP does not bind positively charged DEAE resins but might indirectly interact with DEAE resins through those impure proteins. To further confirm this hypothesis, capillary electrophoresis (CE) was applied to examine the net surface charge of HP-NAP. The net surface charge of HP-NAP was neutral at pH 7.0 to 8.0 and both neutral and slightly negative at pH 8.5 and 9.0. Interestingly, the purified recombinant HP-NAP from gel filtration was capable of binding DEAE Sepharose reins at pH 7.0 to 9.0, and only negative surface charges of the purified recombinant HP-NAP was detected by CE at pH 8.5 and 9.0. Thus, different electronic states of recombinant HP-NAP are purified through ion-exchange purification and gel filtration. The net surface charge carried by HP-NAP might be the main factor for determining whether HP-NAP binds DEAE resins. However, the charge distribution on the surface of HP-NAP and DEAE resins also play an important role for the interaction of HP-NAP and DEAE resins during the negative purification.