| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 90 === Helicobacter pylori, an important human gastric pathogen, infects about one half of the world’s population. In addition to being the cause of chronic gastritis and ulcer, the pathogenic bacteria is associated with gastric adenocarcinoma and lymphoma. Fucosyltransferase are the enzymes that catalyze the transfer of fucose to different acceptors for the biosynthesis of Lewis carbohydrate determinants, Lea, Leb, Lex, Ley, sLea and sLex, which exist on the surface of mammalian cells.
H. pylori expresses Lex antigen to avoid the detection of host’s immune system. Such mimicry also plays an important role in mucosal adhesion, immune evasion, and auto-antibody induction. To investigate the corresponding fucosyltransferases in detail will help us understand further the relationship between Lewis antigens and pathogenesis. We have cloned the a1,3-fucosyltransferase (a1,3-FucT) from the H. pylori CCRC 17026. The recombinant protein was overexpressed in E. coli BL21 (DE3) under T7 promoter control. The enzyme activity was observed by the formation of Lex on TLC. The fluorescence-labeled acceptors, e.g. pyridylaminated (PA-)sugars, were also applied for the detection of enzyme activity. The enzymatic products were separated and quantified by normal phase HPLC and confirmed by 1H NMR and Mass analysis. Most expressed FucT was pelleted down at 20,000 g during differential centrifugation, but the enzyme activity was found to be predominant at this fraction. Although the soluble enzyme was not obtained, we could partially purify the FucT by differential centrifugaion. Polyclonal anti-FucT-His6 antibodies were generated by subcutaneous injections of the protein to rabbits. The resulting antibodies were found to be able to detect 2 ng FucT in a 1:10000 dilution. Finally, we have prepared the C-terminally truncated FucT with 28 and 38 fewer amino acids, both proteins could be expressed as soluble forms and purified by immobilized metal affinity chromatography. Their specific activity was observed to be 1000-fold highe
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