- 學位論文
胃幽門螺旋桿菌之麩胺基硫亞精胺合成酶(HP0233)的表現質體建構及功能分析
Molecular Cloning and Functional Characterization of Glutathionylspermidine Synthetase (HP0233) from Helicobacter pylori
摘要
HP0233有大於20%的蛋白質胺基酸序列和其他物種的GspS相同，例如：大腸桿菌。將1173鹼基對的HP0233基因建構到pQE30載體中並且在大腸桿菌SG13009菌株中作大量蛋白質表現。在經過蛋白質純化後，一升菌液可得到20毫克並且N端帶有六個組胺酸的重組蛋白質。接著利用HP0233重組蛋白質製備多株抗體，藉著西方點墨法可在不同生長條件下辨認出單一條帶。經過質譜儀和超高速離心法分析後，指出HP0233重組蛋白質以單體存在於水溶液中。利用圓二色譜光譜儀分析指出其蛋白質主要二級結構在PH值5到10之間是穩定不變的，而在熱變性中得到蛋白質對熱的耐熱度約為53 ℃。 在蛋白質功能性的相關研究結果得到HP0233在37 ℃和含有5 mM MgCl2、pH值8的反應溶液中具有ATPase的活性，在pH值為8時，ATPase活性最高。當HP0233蛋白質存在於45 ℃二十分鐘後，加入反應溶液中在37 ℃反應，發現其酵素活性有下降現象。其ATPase動力學的相關參數也已經決定：Km值為0.48±0.16 mM；kcat值為1.34±0.13 min-1。在濃度為5 mM的二價陽離子鈷、錳、鐵、鈣皆可以取代鎂離子供給ATPase酵素活性，但是效果沒有鎂離子好。在加入濃度0.5到3 mM的ADP於反應溶液中，ATPase活性會被ADP所抑制。 至於synthetase活性目前尚未明顯的被偵測出來，接著會利用抗體將胃幽門螺旋桿菌中的內生性HP0233蛋白質進行純化，並且將synthetase酵素活性實驗的條件更理想化後再進行實驗分析。另外，當胃幽門螺旋桿菌存在於酸性環境時，HP0233的蛋白質表現量較低。
並列摘要
The deduced amino acid sequence of the HP0233 gene shares more than 20% sequence identity with most glutathionylspermidine synthetase from several species, such as E. coli. The HP0233 open reading frame (1173 bp) was cloned into the pQE30 vector and over-expressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0233 protein was purified by Ni–NTA affinity chromatography at a yield of 20 mg/L of bacteria culture. The recombinant HP0233 protein was used to produce polyclonal antibodies which could recognize single band of protein in H. pylori at different grown conditions by Western blotting analysis. Mass spectrometry and analytical ultracentrifugation have shown that the recombinant HP0233 protein exists as a monomer in solution. The major secondary structure was stable in the range of pH between 5.0 and 10.0. Thermal unfolding transition showed Tm valve was about 53 °C by circular dichroism spectroscopy. Results from functional assays showed that HP0233 possessed ATPase activity in reaction buffer containing 5 mM MgCl2 at pH 8.0 and 37 °C. The highest ATPase activity of HP0233 protein appeared in reaction buffer at pH 8.0. Reduced enzyme activity was observed in Hp0233 protein after stored at 45 °C for 20 min and assayed at 37 °C. The kinetic parameters of the recombinant HP0233 for the ATPase activity have been determined to have the apparent Km value of 0.48±0.16 mM for ATP substrate and a kcat value of 1.34±0.13 min-1. At 5 mM of Co2+, Mn2+, Fe2+, and Ca2+ can substitute for magnesium in Mg2+-dependent ATPase, but they were not able to significantly contribute to the enzymatic activity. ADP inhibition at 0.5-3.0 mM would suppress ATPase activity of HP0233 protein. The synthetase activity of HP0233 protein was not yet detected significantly. The endogenous HP0233 will be collected from H. pylori lysate with antibodies and the assay condition for synthetase activity needs to be further optimized. In vivo, HP0233 expression was reduced when H. pylori in acidic environment.