Cloning, expression, and purification of HP1286

The construct for expression of sequence encoding amino acids 18 to 182 of HP1286 was generated by cloning of the PCR product at NdeI and XhoI sites of the vector pET28b+ (Novagen). The genomic DNA from H. pylori 26695 was used as template in the PCR reaction. Primers used were: 5′-GCGAGCTCATATGAAACCTTATACGATTG-3′ (Forward) and 5′-AAACTCGAGTTGGGCGTAAGCTTCTA-3′ (Reverse). Induction of the protein was carried in E. coli BL21 (DE3) at 37°C for 3–4 h with the addition of isopropyl thio-β-d-galactoside (100 μM) in Luria-Bertani (LB) media. Hexa-His-tagged HP1286 was purified from the soluble fraction by chromatography on Talon resin (Clontech). To remove imidazole, the purified protein was dialyzed overnight followed by another purification step using an endotoxin removal column (Pierce). Amount of endotoxin in the purified protein was assessed by an endotoxin detection kit (Pierce) and the purified protein was concentrated using Amicon Ultra-4 centrifugal filter (Millipore). After staining with the Coomasie brilliant blue dye, the proteins appeared as a single band under reducing conditions. For the generation of an irrelevant-His-tagged (Irr-His) protein, another H. pylori gene (HP1563) was cloned in the same vector pET28b+ at Nde1 and XhoI sites. Irr-His was overexpressed in E. coli BL21 (DE3) and purified from the soluble fraction. The purified protein was of expected size (24 kDa).