利用液相層析質譜儀鑑定大腸桿菌胞內去氧核醣核酸水解酵素

Abstract

中文摘要 大腸桿菌（Escherichia coli）是目前研究最為透徹的格蘭氏陰性菌（gram negative bacteria），其基因體庫也因定序技術的進步在二十世紀末建立，更隨著分子生物學的進展成為了表現重組基因技術中最為大眾所利用的工具。本實驗室致力於去氧核醣核酸水解酶的研究中也多利用大腸桿菌作為表現重組去氧核醣核酸水解酶基因以及定點突變法的工具。但是在表現相對活性較低的重組去氧核醣核酸水解酶時，大腸桿菌內生的核酸水解酶卻也影響了重組蛋白活性的偵測以及生化特性分析時的操作，因此本篇論文利用本實驗室最常用作表現蛋白之宿主大腸桿菌株BL21 (DE3) pLysE作為實驗的材料，嘗試結合液相層析管柱純化內生性蛋白質，並利用液相層析質譜儀搭配對NCBI大腸桿菌基因體庫作搜尋的方式來鑑定，大腸桿菌胞內造成影響重組去氧核醣核酸水解酶活性的主要核酸水解酵素為何。 將培養好的菌液經過冷凍解凍法破菌並離心取得上清液後，經過DEAE-Cellulose、Ceramic Hydroxyapatite、Phenyl-Sepharose、Mono Q 5/5 HR等四根層析管柱的分離純化過程後，利用真空乾燥法減少樣品體積後利用SDS-PAGE電泳的技術並以活性染色法與Coomassie blue染色法等兩種不同染色法去確認活性染色膠片中在UV光照下28 kDa附近產生因為水解DNA而造成的暗帶，而後將Coomassie blue染色膠片的相對應位置之蛋白質色帶切下，經Trypsin digestion後進行LC-MASS的鑑定分析，由該蛋白質色帶所鑑定出可能目標蛋白質之中有一為Endonuclease I precursor符合實驗的目標。查閱文獻資料顯示﹕Endonuclease I是一種會被雙股transfer-RNA強烈抑制水解DNA能力，主要分布於膜間帶（periplasma）的去氧核糖核酸水解酶。是以利用Sucrose-EDTA法將存於膜間的酵素抽出後與胞內酵素的活性以SDS-PAGE的活性染色法相比較，可以由主要影響的酵素座落的暗帶說明其相對分子量與經由層析管柱純化後的活性沖提液都相同，而利用RNA inhibition的實驗證明經過四根層析管柱純化後的樣品其水解DNA能力有明顯被抑制現象，這也直接的證明了存在於大腸桿菌胞內並對重組去氧核醣核酸水解酶活性偵測造成影響的酵素是以往認為存於膜間帶或是分泌出胞外的Endonuclease I。

Abstract Escherichia coli was the most well-studied gram-negative bacteria and has been wildly used as a useful tool in the field of molecular biology and gene recombination technology. The genome of the bacteria had been sequenced at the end of the twentieth century after the development of modern gene sequencer. The established E. coli genome database provided an instant tool for protein and gene identification. Our laboratory has been devoted in the research of deoxyribonuclease, whereas E. coli was often used as the tool to express the recombinant proteins for the mutated genes. The endogenous nuclease with the activity to hydrolyze nucleic acids might interfere with the detection of the recombined or mutated deoxyribonulease with low activities. The phenomenon interfered the manipulation of experiments. Thus, it made us to investigate as which enzyme that were making the interference. By using the liquid chromatography purification system, and the modern technology of the LC-MS spectrometry, we were able to identify an endogenous E. coli nuclease that often interfered our results. The cultured bacteria were pelleted down by centrifugation, and disrupted after the freeze and thaw procedure followed by sonication. The supernatant fraction was obtained by centrifugation to remove the bacteria debris. The sample were partially purified by four consecutive liquid chromatography columns, ( DEAE-Cellulose, Ceramic hydroxyapatite, phenyl-Sepharose and Mono Q 5/5 HR ). Fractions with nuclease activitywere collected and lyophilized to reduce their volume. The collected sample were dissolved in SDS-PAGE sample buffer, and were subjected to SDS-PAGE analysis. The gel was stained with activity staining and Coomassie blue staining. The zymogram showed that a dark band with nuclease activity around molecular-weight of 28 kDa. The protein band located at the corresponding position on the coomassie blue stained gel was cut, and subjected to in-gel trypsin digestion. The eluted protein were than used for LC-MS analysis. The analysis of spectrometry provided 15 protein candidates that have been analyzed in the sample. E. coli endonuclease I precursor was the one which matched the biological characters. Endonuclease I of E. coli is a periplasmic DNase readily inhibited by different RNA species. To determine the bacterial lysate that containd the enzyme as the one in periplasmic fractions, the sucrose-EDTA method was used. The zymogram analysis showed that the most abundant nucleolytic enzyme in bacterial lysate, periplasmic fraction and partially purified fractions were of same molecular weight. The result of RNA inhibition assay showed that the nucleolytic activity of the purified sample were obviously reduced while incubated with yeast t-RNA, These results provided a direct evidence that the nucleolytic enzyme in purified sample was E. coli endonuclease I.