Identification and Structural Characterization of a 10k Hypothetical Protein Expressed Differently in Two Strains of Helicobacter pylori by a Proteomic Approach

• Main Authors: Lo, Su-Tang, 羅旭棠
• Other Authors: Shyh-Horng Chiou
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/52339930524306892897

碩士 === 國立臺灣大學 === 生化科學研究所 === 91 === Helicobacter pylori is an important human gastrointestinal pathogen for which the entire genome sequence is known. This microorganism displays a uniquely complex protein expression pattern and expresses a variety of enzyme and adhesion molecule to interact with human gastric niches, and survives in the strong acidic environment of human stomach. The identification and characterization of proteins expressed by H. pylori is a prerequisite for the development of vaccines designed to interfere with bacterial colonization of host tissues. In this study we have employed a proteomic approach to investigate the protein expression profiles from two different H. pylori strains isolated from patients with gastric cancer and duodenal ulcer. Based on high-resolution two-dimensional gel electrophoresis coupled with MALDI-TOF mass spectrometry, and NCBI database searches, several protein spots were found to be expressed differently in these two strains of H. pylori. Further, through peptide mass fingerprinting by in-gel spot digestion revealed one heretofore unknown hypothetical protein located at an isoelectric point of 8.7 and with a molecular mass of about 10 kDa, herewith designated as Hyp10. We have cloned the ORF (open reading frame) HP0495 and transformed into E. coli system for the expression of this protein. The recombinant Hyp10 was separated and purified via a cation-exchange chromatography coupled with a Millipore membrane filtration. Bioinformatics was also used to analyze and compare the domain structure of Hyp10 protein with other proteins in the databank. The secondary structure of Hyp10 was analyzed by circular dichroism coupled with structure prediction based on protein sequence. GST pull-down assay was employed to study the protein-protein interaction between Hyp10 and other proteins in H. pylori. Preliminary crystallization of Hyp10 for x-ray diffraction and NMR study in solution are currently in progress.