Recombinant Hp1018/19Δsp Protein Purification

The construct Hp1018/19Δsp was amplified from genomic DNA of H. pylori strain 26695 using the primers 5′-aaggatccggcaatatccaaatccagagcatg-3′ and 5′-aagaattcgacccacccctatcatttcacc-3′ with Pfx DNA polymerase in supplied buffer with 2× PCR Enhancer (Invitrogen). The amplified BamH1/EcoR1 flanked PCR product was then ligated into the pGEM-T Easy plasmid (Promega), subcloned into the pGEX-6P-1 plasmid DNA (GE Healthcare Life Sciences) and transformed in E. coli BL21. The construction of the protease-inactive Hp1018/19Δsp^S205A protein, serine 205 was mutated to alanine using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. For heterologous overexpression and purification of GST-Hp1018/19Δsp, transformed E. coli was grown in 500 ml TB medium to an OD₅₅₀ of 0.6 and the expression was induced by the addition of 0.1 mM isopropylthiogalactosid (IPTG). The bacterial culture was pelleted at 4000×g for 30 minutes and lysed in 25 ml PBS by sonification. The lysate was cleared by centrifugation and the supernatant was incubated with glutathione sepharose (GE Healthcare Life Sciences) at 4°C over night. The fusion protein was either eluted with 10 mM reduced glutathione for 10 minutes at room temperature or cleaved with 180 U Prescission Protease for 16 h at 4°C (GE Healthcare Life Sciences). Elution and cleavage products were analyzed by SDS PAGE and zymography.