- 學位論文
胃幽門螺旋桿菌脫氧尿嘧啶三磷酸核苷酸水解酶的表現質體建構以及功能分析
Molecular cloning and functional characterization of deoxyuridine 5’triphosphate nucleotide hydrolase from Helicobacter pylori
摘要
脫氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)能夠催化脫氧尿嘧啶三磷酸(dUTP)水解成脫氧尿嘧啶核苷酸(dUMP)及無機焦磷酸(PPi)，這反應能防止細胞內uracil被併入雙股的DNA當中，它是個參予核酸代謝的酵素。關於核酸代謝的酵素已經被當作一些疾病藥物研發的目標，近來的研究指出脫氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)也包含在這些藥物研發的目標酵素之中。胃幽門桿菌內脫氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)的結構以及酵素動力學上的功能分析，對於發展對抗此酵素的藥物是個先決的條件。 在TIGR的生物資訊網站上，Hp0865被預測為胃幽門桿菌內脫氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)的轉錄基因。將此Hp0865基因建構到pQE30載體中並且在大腸桿菌SG13009菌株中進行大量蛋白質表現。一公升的菌液可以得到將近25毫克的蛋白質。重組蛋白Hp0865儲存在Eluting buffer (20 mM Tris-HCl, 300 mM NaCl, 200 mM imidazole, pH 8.0)裡並不會有蛋白質沉澱的現象發生。SDS-PAGE的結果顯示出重組蛋白Hp0865分子量為17 KD，此外超高速離心顯示此蛋白是個三聚體。將重組蛋白Hp0865當作抗原注射入兔子誘發免疫反應，產生多株抗體。所得到的多株抗體能使用在偵測胃幽門桿菌內Hp0865的蛋白質表現量。經由抗體的使用，在酸的處理以及不同時間點的觀察，胃幽門桿菌體內的脫氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)表現量並沒有明顯的變化。 由dUTPase assays以及定量無機焦磷酸(PPi)的結果可得知在pH 8.0以及攝氏25度的條件下，5微克重組蛋白Hp0865在7分鐘內能夠水解掉幾乎全部的60 uM dUTP。從酵素活性的的結果可得知:反應溶液中有1 mM Mg2+時，Hp0865蛋白質動力學參數Km和Kcat分別是0.36 uM以及0.7 s-1；此外產物無機焦磷酸(PPi)對於反應有著競爭型抑制的作用，其抑制常數(Kip)為11.8 uM。 另一產物脫氧尿嘧啶核苷酸(dUMP)其抑制作用甚小並不足以構成產物抑制作用。化學藥物NaF和Resveratrol對於重組蛋白Hp0865並沒有任何影響。 經由不同核苷酸的水解測試，重組蛋白Hp0865對於dUTP呈現很高的專一性。在4oC儲存一個月之久的重組蛋白Hp0865仍保有~78%的活性。此蛋白即使前處理60 oC，15分鐘仍有著一半的活性。此外酵素活性在pH 7至pH 9.5 保持良好的催化效率，在pH 7.5時酵素展現的活性較高一些。酵素活性隨著鎂離子的濃度(0.1-1 mM)上升而增強。在相同濃度之下甚至可以發現鈷和鋅對酵素活性的增加效果較好。重組蛋白Hp0865在不同離子的處置之下，經由CD在波長200-240 nM這範圍的測定，可以發現明顯的波長變化。 無機焦磷酸(PPi)在低濃度時對於重組蛋白Hp0865的產物抑制作用以及鈷離子和鋅離子能夠取代鎂離子給予酵素更佳的活性是兩個新發現。
並列摘要
Deoxyuridine 5’-triphosphate nucleotide hydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. It is an enzyme in the pyrimidine- nucleotide metabolism. Enzymes in the DNA metabolism have been used as targets for drugs against cancer and virus-infected diseases. Recent investigations have shown that dUTPase might be included among these targets. Characterization of dUTPase of H. pylori strain 26695, structurally and kinetically, would be a prerequisite for the design of such drugs. The Hp0865 gene encoding dUTPase in H. pylori strain 26695 was cloned into the pQE30 vector and overexpressed in E. coli strain SG13009. The resulting rec-Hp0865 protein with N-terminal 6x-His-tag was purified by Ni-NTA column at a yield of 25 mg/L of bacteria culture. The Rec-Hp0865 protein stored in eluting buffer (20 mM Tris-HCl, 300 mM NaCl, 200 mM imidazole, pH 8.0) would not aggregate. The molecular weight of rec-Hp0865 was determined to be 17 KD by SDS-PAGE. A trimer structure in native protein was suggested from the results of a major peak at 51 kDa characterized by the analytical ultracentrifugation. Purified rec-Hp0865 protein as antigen was used to produce polyclonal antibodies which would detect endogenous Hp0865 protein level in H. pylori. No change of endogenous Hp0865 protein level was observed from bacteria under acidic stress or in different culture times . Spectrophotometric assays showed that 5 ug rec-HP0865 protein would hydrolyze most of 60 uM dUTP at 7 min in reaction buffer including 1 mM MgCl2 at pH 8.0 and 25 oC.. The determination of released PPi by colorimetric assay revealed that 94.6 % dUTP was hydrolyzed in above mentioned condition. The kinetic parameters of this dUTPase have been determined to have a Km of 0.4 uM and a kcat of 0.7 s-1 for dUTP substrate in the presence of 1 mM Mg+2. The product, PPi, causes competitive inhibition with a Kip value of 11.8 uM.. Another product, dUMP, has less inhibition than PPi on enzyme activity. In addition, neither NaF (phosphatase inhibitor) nor resveratrol affected the enzymetic activity of rec-Hp0865. The enzymetic activity of rec-Hp0865 was highly specific for dUTP as substrate. The rec-Hp0865 protein could display ~78% dUTPase activity after one month. Reduced enzyme activity was observed in Hp0865 after pre-incubation at above 60 oC for 15 min. The enzyme activity was not sensitive to pH within the range (pH 7 - 9.5). Dose-dependent increase on enzyme activity appeared in reaction containing 0.1-1 mM MgCl2. The enzyme showed much activity in the presence of Co2+ or Zn2+ instead of Mg2+ at the same concentration. CD spectra changes in the 200–240-nm wavelength range were observed by the substitution of Mg2+ to Zn2+ or Co2+ at a 1 mM. The product inhibition of PPi at low concentration and Zn2+, Co2+ preference instead of Mg2+ for dUTPase activity are novel discovery for rec-Hp0865 protein.
參考文獻
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