Abstract
Helicobacter pylori is a Gram-negative bacterium that colonizes the stomachs of an estimated half of all humans. In the absence of antimicrobial therapy, H. pylori maintains residency in the stomach for the remainder of its host’s lifespan. In a subset of humans, H. pylori infection leads to serious disease such as duodenal or gastric ulcer, gastric adenocarcinoma, or mucosa-associated lymphoid tissue lymphoma. It was also found that H. pylori can enhance gastric epithelial cell proliferation and attenuate apoptosis in vivo, which may partially explain the increased risk of gastric cancer associated with this disease. During the past years, a great deal of progress has been made in defining genetic variation among H. pylori isolates. The tools and approaches used to address this problem illustrate how the current revolution in genomics is affecting the study of microbial pathogenesis. The genomes 26695 strain were sequenced in their entirety, 26695 having been isolated from a patient with gastritis. Although lots of efforts have been made on the studies of genomics of H. pylori, little has been done on the proteomics especially structural genomics studies of H. pylori.We have accomplished the following work for this project: Twenty of the H. pylori genes were chosen for structural studies.These genes are HP-1144, HP-1215, HP-1423, HP-0892, HP-0496, HP-0495, HP-0274, HP-0902, HP-0199, HP-0817, HP-0222, HP-0385, HP-1049, HP-1219, HP-1324, HP-1425, HP-1492, HP-0032, HP-0458, and HP-0187. The PCR products could be amplified in the presence of 1-10 ng genomic DNA template at annealing temperatures of: 57 oC - HP-1144, HP-1215, HP-1423, HP-0892, HP-0496, HP-0495, HP-0222, HP-0385, HP-1049, HP-1219, HP-1324, and HP-1425; 53 oC - HP-0274, HP-0902, HP-0199, HP-0817, HP-1492, HP-0032, HP-0458, and HP-0187. We are subcloning these targets into pET22b and HP-0495, HP-0222, HP-0385, HP-0187, HP-1492, HP-0458, HP-1324 was expressed successfully .Now Hp-0385, HP-0495, HP-1492, HP-0458, were purified.CD may provide a useful secondary screen in structure proteomics. From the CD spectra,we can better understand the contribution of protein stability to sample behavior.Thermal melting of HP-0385, HP-0495,in phosphate buffer at PH6.5 indicated unfolding of the secondary structure with Tm of 70.08oC,85.72oC, suggests that HP-0495, HP-0385, exhibits high thermostability. Urea denaturation suggests that HP-0495 exhibits high structural stability with a Cm of 6.18M. We screen HP-0385 by 1H-15N HSQC which may aggregation in high concentration. Finally, we selected HP-0495 and carried out using 15N/13C double labeled proteins on our 500 and 600-MHz NMR spectrometers. The backbone sequential assignments were determined from six triple resonance experiments (HNCA, HNCO, HN(CO)CA, HN(CA)CO, CBCANH, CBCA(CO)NH).