Summary auto-generated
This article investigates the periplasmic protease HP1084 from Helicobacter pylori, a gram-negative bacterium. The researchers identified and characterized HP1084 as a serine protease using molecular and biochemical methods. They cloned the hp1084 gene, expressed the recombinant protein, and analyzed its structural features, including signal peptides and predicted cleavage sites. The study demonstrates that HP1084 exhibits protease activity against various substrates and localizes to the periplasm. The protease cleaves proteins with specificity for certain amino acid sequences. The authors compared HP1084 to other characterized H. pylori proteases and examined its potential role in bacterial pathogenesis. Expression levels were analyzed under different growth conditions. The research indicates HP1084 may contribute to H. pylori virulence through protein degradation in the periplasmic space. The findings provide insights into H. pylori proteolytic mechanisms and potential targets for therapeutic intervention against this important human pathogen.
Key findings
- HP1084 is a periplasmic serine protease from Helicobacter pylori with specific substrate cleavage patterns
- The protease contains an N-terminal signal sequence directing it to the periplasm and a predicted catalytic serine residue
- HP1084 expression varies under different growth conditions, suggesting regulation in response to environmental stimuli
- The enzyme demonstrates proteolytic activity against multiple substrates with sequence specificity
- HP1084 may contribute to H. pylori virulence and pathogenesis through periplasmic protein degradation
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Abstract
The use of alkaline phosphatase fusion methodology to identify Helicobacter pylori exported proteins enabled the identification of an open reading frame (ORF) encoding a highly immunogenic, previously uncharacterised exported protein. The predicted amino-acid sequence displays a typical N-terminal signal peptide and contains regions of C-terminal hydrophobicity consistent with a membrane-associated protein. Southern blot analysis revealed that the gene encoding the protein was absent in several Helicobacter spp. and a combination of PCR and sequence analysis of the amplified gene showed that it is highly conserved amongst isolates of H. pylori. To obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a ß-galactosidase (ß-gal) fusion in Escherichia coli and the protein was purified by affinity chromatography and proteolytic cleavage of the ß-gal portion. The purified protein, which has an apparent mol. wt of 18 kDa, was recognised by antibody present in 71% of sera from patients infected with H. pylori, but in only 16% of sera from patients with unrelated or no gastrointestinal disease, by Western blot assays. These results indicate that the 18-kDa protein from H. pylori is immunogenic and is expressed in vivo.