Abstract
Helicobacter pylori is considered the most common infectious agent among humans worldwide. Recently, the neutrophil-activating protein (NAP), a H. pylori protein that is highly immunogenic in human, has been identified. However, purification of NAP from H. pylori is difficult due to its fastidious growth in microaerophilic condition and poor yield. To date, it has never been reported that the functional recombinant NAP were purified from E. coli expression system. To determine whether the functional dodecameric NAP could be obtained from E. coli expression system, both recombinant native and His-tag fusion NAPs expressed in E. coli were purified and characterized. By size exclusion and analytical ultracentrifuge analyses, both recombinant native and His-tag fusion NAPs are assembled into multimeric forms. By circular dichroism analysis, the secondary structures of both purified recombinant NAPs were identified as the typical a-helix. By luminol-enhanced chemiluminescent assay and westen blotting, both the recombinant native and His-tag fusion NAPs were able to cause the O2.- release from cells and increase phosphorylation of extracellular regulated kinase (ERK). From my E. coli expression system, a large amount of recombinant NAPs were obtained. Multimeric assembly and activity of both recombinant native and His-tag fusion NAPs are similar to those of native NAP from H. pylori. The results indicate that the recombinant NAPs obtained from our E. coli expression system is functionally active. Importantly, this E. coli expression system can provide a lot of recombinant NAPs for basic studies, vaccinal development, or drug design.