篩選噬菌體P1201強勢啟動子俾應用於構築Corynebacterium glutamicum之表現載體

Corynebacterium glutamicum is generally recognized as nonhazardous organism, which is safe to handle. Based on its extremely well investigated central metabolism and well-established molecular biology tools, C. glutamicum may be very suitable as an organism for the expression of heterologous gene in high (G+C) bacteria. Promoter regions from putative ORFs of corynebacteriophage P1201 were cloned into 5' end of reporter cat gene and then transformed into C. glutamicum. Promoters from the genes corresponding to ORF 24 and ORF 29 showed CAT specific activities of cat enzyme was 7.17 and 8.17 U/mg protein, repectively. DNA fragments of Sau3AI digested corynephage P1201 genomic DNA were also cloned into pORC, a promoter probed vector, with cat as a reporter gene. P'1,P'2,and P'3 fragments show cat enzymes with specific activities of 8.67, 6.28 and 3.50 U / mg protein repectively, were obtained which are higher than that of trc promoter from E.coli and gdh promoter from C. glutamicum glutamate dehydrogenase gene. Corynephage P1201 structural proteins including portal protein, tail protein, and head protein were found by SDS－PAGE analysis, LC/MS/MS analysis, and N－terminal amino acid sequencing. Putative head protein from ORF 39 exhibited different mobility in SDS－PAGE analysis indicating that proteolytic cleavge might be involved in the maturation of phage capsid protein.由於 Corynebacterium glutamicum 被認定為安全的微生物，在工業操作上相當的方便。而且其代謝調控已有深入研究，基因操作系統也已建立，此菌可能非常適合使用於高 (G+C) 外源基因之大量表現。本研究選殖glutamate dehydrogenase 基因 (簡稱gdh)之啟動子區及trc啟動子於報導基因cat的 5’ 端，並轉形至 C. glutamicum ，在 CM broth 的培養條件下，發現trc之啟動子表現最好，cat之比活性為6.22 U / mg protein，gdh啟動子Cat比活性為3.94 U / mg protein。預測可能表現基因（ORF）的啟動子區域，將用PCR方式增幅啟動子，將其構築入啟動子偵測載體pORC並以電轉形法轉形入 C. glutamicum LS1183，在 CM 平板上（含有 chloramphenicol 5 ug / ml）進行篩選，發現ORF 24與ORF 29的啟動子活性最佳，其比活性分別為 7.17及 8.17 U / mg protein，較trc強勢啟動子活性提升約30％。將corynephage P1201 DNA 以 Sau3AI 剪切，並選殖入啟動子偵測載體pORC，得到P′1啟動子其表現的cat比活性為8.67 U/mg protein，比目前使用的Ptrc強勢啟動子提升約40％。P′2啟動子其表現的cat比活性為6.28 U/mg protein、P′3啟動子其表現的cat比活性為3.50 U/mg protein，而在P′2與P′3啟動子上游加入不同DNA片段的質體，所表現的cat比活性比單獨具有P′2或P′3啟動子的質體提升一到兩倍。利用LC/MS/MS方法鑑定噬菌體P1201的結構蛋白身份，得到band A比對到ORF 28，推測為噬菌體P1201的portal protein；band D、E、F、G、K、M、N比對到ORF 39推測為head protein；band J、K、N比對到ORF 45推測為噬菌體P1201的tail protein；band O比對到ORF38推測為head protein，proteolytic cleavage可能導致 ORF 39在SDS-PAGE中有高度的移動性。中文摘要.................................................i 英文摘要............................ ii 目錄.................................................iii 圖目錄………………………………………………...….……..vi 表目錄…………………………………………….. ..…….……vii 縮寫對照表…………………………………………………..…... vii 一、前言………………………………………………....……………1 （一）Corynebacterium ssp. 表現系統之簡介…………………..……………1 （二）啟動子在基因表現所扮演的角色.................................2 1.啟動子............................................2 2. mRNA的穩定性..............................3 （1）RNase 的作用......................3 （2）RNA的二級結構..................4 3.核醣體結合位置（ribosome-binding site , RBS）........................................4 （三）研究動機與策略............................5 （四）研究的目的..................................7 二、材料與方法................................................8 （一）藥品.............................................8 （二）菌種、噬菌體、質體及培養基..............................8 （三）引子 (Primer) 的設計.........................8 （四）質體抽取.........................................8 （五）質體的轉形作用..........................8 （六） DNA 回收、接合及聚合酵素連鎖反應.....................................8 （七）噬菌體 DNA 的抽取..................9 （八）噬菌體結構蛋白的抽取................9 （九）蛋白質電泳分析...........................10 （十）利用LC/MS/MS方法鑑定噬菌體P1201的結構蛋白身份....................10 （十一）利用穿透式電子顯微鏡（analytical electron microscope）觀察噬菌體P1201………………………………………………………..……10 （十二）噬菌體 P1201 可能的啟動子序列的分析........................................ 10 （十三）蛋白質濃度的測定................... 10 （十四）報導基因Cat（chloramphenicol acetyltransferase）表現強度之平板分析法....................................10 （十五）報導基因Cat之酵素活性分析...............................................11 三、結果...................................................13 （一）利用gdh及trc 基因的強勢啟動子表現Cat基因...................................13 （二）預測噬菌體P1201可能表現基因的啟動子區域....................................13 （三）由 P1201 DNA 分離具有啟動子功能的DNA片段..............................14 （四）P1201噬菌體強勢啟動子之組合對於啟動子活性影響........................14 （五）噬菌體 P1201構造蛋白的分析.....................................16 四、討論.......................................19 （一）噬菌體所篩選啟動子之強勢表現.......................19 （二）啟動子的上游序列對於啟動子表現強度的影響.......19 （三）P1201噬菌體所篩選強勢啟動子的序列分析.......19 （四）噬菌體P1201的結構蛋白在SDS–PAGE的高移動性........20 五、總結....................22 六、參考文獻...............................4