Abstract
In the last few years, genome sequencing projects have produced a wealth of information on the organization of living organisms. The flood of sequence information coupled with recent advances in molecular and structure biology have led to the concept of “structural proteomics” or “structural genomics”, the determination of three-dimensional protein structures on a genome-wide scale. An important use of three-dimensional structural information of proteins is to uncover clues as to a protein’s functions that are not detectable from sequence analysis. In this component project, we will use multi-dimensional NMR techniques to determine the three-dimensional solution structures of novel proteins (molecular weight □ 20 kDa) from the human pathogenic bacteria Helicobacter pylori. H. pylori is a micro-aerophilic Gram-negative, slow-growing, spiral-shaped and flagellated bacterium that colonizes the stomachs of an estimated half of all humans. (30% of the population in developed and up to 90% of the population in developing countries.) In a subset of humans, H. pylori infection leads to serious disease such as peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma or gastric adenocarcinoma. The target chosen for NMR screening are based on the following characteristics: (1) no sequence homologue in the PDB database (2) no predicted transmembrane domain (3) molecular weight of single chain polypeptide under 20 kDa. Twenty of the H. pylori genes were chosen for structure 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, HP-0187.The PCR products could be amplified easily in the presence of 1-10 ng genomic DNA template at annealing temperatures of 56℃and 53℃. No unspecific products were found. These PCR products were sub-cloned into pGEM-T (promega) and checked by DNA sequencing. The results of DNA sequencing showed all of the targets were accurate expect HP0199. We are sub-cloning these targets (expect HP0199) into pET-22b for protein expression and purification. Among these target genes, further structural studies of HP-0222 will be carried out using CD spectrometer and NMR spectrometer. Because HP-0222 is acid-induced protein, so I do the CD experiments in different pHs. And NMR condition that I choose is in pH3.5 buffer condition, one-D and HSQC spectrum are the parts of articles. The completion of this component project will not only provide novel protein structures of Helicobacter pylori and its associated proteins, but also help the proteomic studies of the roles of Helicobacter pylori in human diseases.