Abstract
Single strand DNA binding protein (SSB) plays essential roles in many processes related to DNA metabolism such as DNA replication, repair, and homologous genetic recombination. The HP1245 gene was annotated as SSB in Helicobacter pylori strain 26695. However, there are no functional or structured studies for this SSB up to now. The full length (179 residues), three different C-terminal truncated species (106, 122 and 134 residues) and four different site directed mutants (F37A, F50A, F56A and W84A) were individually sub-cloned into pQE30 vector and expressed in E. coli SG13009. After IPTG induction, each species of recombinant (rec) HP1245 protein with N-terminally 6xHis-tagged fusion was purified by Ni-NTA affinity chromatography and its identity confirmed by mass spectrometry and Western blotting analysis using an anti-His-tag monoclonal antibody. Full length and various mutant recHP1245 proteins were homo-tetramer according to mass spectrometry and sedimentation velocity ultracentrifuge analysis, respectively. However, even in NaCl containing buffer, full length recHP1245 protein was easy to degrade after stored at 4℃ for 2 weeks, but C-terminal truncated proteins (122 and 134 residues) were stable for months. This indicated that the inherently disordered C-terminal region of SSB in H. pylori, similar to that in E. coli, may affect protein stability. The ssDNA binding property of HP1245 was performed through electrophoretic mobility shift assays (EMSA) to determine the binding affinity and the binding domain. The result indicated that the affinity of full-length or truncated recHP1245 proteins (122 and 134 residues) with biotin-labeled d(T)35 ssDNA was similar. It meant that the ssDNA binding domain was located at the N terminal. Furthermore, the site directed mutants, F37A, F50A, F56A and W84A were also individually measured by the same method. These results indicated that either F56A or W84A residue of recHP1245 protein would decrease the ssDNA binding affinity. Therefore, the two residues played a crucial role on ssDNA binding. These results suggested that the aromatic residues in this protein might contribute certain roles on ssDNA binding via base stacking interaction with the base in the ssDNA. In order to survive in stomach, Helicobacter pylori must have the ability to modify gene expression in acidic circumstances. To investigate whether HP1245 protein could be acid-induced or not at this stress, H. pylori were individually cultured on Brucella agar plates for 48 h at pH 7.0 and pH 5.5. In this study, the result showed that the protein expression of HP1245 did not have significant difference.