鏈黴菌的端粒連結蛋白與末端蛋白的純化與交互作用

Purification and demonstration of the interaction between TapC and TpgC of Streptomyces.

王萬鈺(Wan-Yu Wang)

指導教授 ： 楊千金

中原大學/理學院/化學研究所/碩士(2011年)

https://doi.org/10.6840/cycu201100977

摘要

摘 要
端粒連結蛋白 (Tap) 與末端蛋白 (Tpg) 在維持鏈黴菌線形染色體或質體中扮演必要的角色，鏈黴菌線形染色體的複製是由中心的複製起點以一般複製方式向兩側進行，當複製進行到末端的時後會在3』端留下缺口，此時需要其他的複製機制去補齊。2003年Bao 和 Cohen利用酵母菌雙雜交法與免疫沉澱法證明鏈黴菌的端粒連結蛋白與末端蛋白具有交互作用，此外他們還用足跡實驗發現端粒連結蛋白會與末端第二、三迴文區結合。由此推論末端的補齊至少需要端粒連結蛋白與末端第二、三迴文區結合並且把末端蛋白帶到末端作為引子進行DNA合成。
本論文分成四部分，第一部分是質體構築，把端粒連結蛋白與末端蛋白的基因分別剪接到蛋白質表現質體pet15，讓表現的蛋白質在N端接上(His)6標識。另外，構築末端蛋白的基因在蛋白質表現質體prsetA，此方式為得不含(His)6標識的末端蛋白。第二部分為蛋白質表現及純化，首先尋找表現條件，例如：培養的溫度、IPTG的濃度和轉速，以求得最佳條件得到可溶性蛋白。再利用鎳金屬親和性層析管柱純化接有(His)6標識的端粒連結蛋白與末端蛋白。第三部分活性測試，我們把純化出來的端粒連結蛋白與末端蛋白進行單股末端序列補齊的實驗，結果顯示端粒連結蛋白不僅讓末端蛋白接上第一個核苷酸，還能延伸數個核苷酸。為排除大腸菌去氧核醣核酸聚合酶(DNA polymerase I)的汙染，我們藉由增加沖洗管柱的緩衝溶液體積或改變緩衝溶液中咪唑(imidazole)和氯化鈉的濃度進行純化。第四部分交互作用，我們利用鎳金屬離子親和性層析管柱的方式，把具有(His)6標識的端粒連結蛋白先固定在樹脂上，再用來純化不含(His)6標識的末端蛋白。結果顯示在0.1M氯化鈉緩衝溶液的條件下，端粒連結蛋白與末端蛋白有交互作用，在0.5M 氯化鈉緩衝溶液的條件下沒有交互作用。

關鍵字

鏈黴菌 ； 端粒連結蛋白 ； 末端蛋白

並列摘要

Abstract
Telomere associate protein (Tap) and terminal protein (Tpg) of Streptomyces are important for maintaining linear chromosomes of Streptomyces. The replication of linear chromosomes of Streptomyces proceeds from central origin of replication to both sides. The replications proceeding to the ends are not completed. It will leave a gap at 3』 end which needs a different machinery to patch. In 2003, Bao and Cohen reported the interactions between Tap and Tpg of Streptomyces lividans by using yeast two-hybrid assay and immunoprecipitation experiments. Besides, they used footprinting analysis to discover that TapC binds DNA end at palindrome II and III. It suggested that the end-patch at least needs TapC binding the II and III palindrome sequence and recruiting TpgC to synthesize DNA.
My research includes four parts. First, cloning plasmids. We cloned the Tap gene and the Tpg gene into expression protein vector, pet15, to tag (His)6 to the N-terminal of the expressed proteins. To get wild-type terminal proteins, the gene was engineered into prsetA instead. Second, expressing and purifying proteins. We first searched the best conditions, for example: culturing temperatures, concentrations of IPTG and rotation rate to get suitable amounts of soluble form of proteins, and then purified the (His)6-TapC and (His)6-TpgC by means of immobilized metal ion affinity chromatography (IMAC). Third, examining the activity of proteins. The results indicated that TapC and TpgC not only tagged the first nucleotide to TP but also extended several nucleotides away. To exclude the possibility of contamination of E.coli DNA polymerase I, the sample was washed more thoroughly at the washing step by increasing the volume of washing buffer and the concentration of imidazole and sodium chloride. Fourth, interaction of TpgC and TapC. (His)6-TapC was first bound on resins and then used to purify the TpgC. The results indicated that the interactions happened in 0.1 M NaCl, but not 0.5 M NaCl.

並列關鍵字

Streptomyces ； terminal protein ； telomere associate protein

參考文獻

1、 Bao, k. and Cohen, S. N. (2001).Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes & Dev.15: 1518–1527

2、 Bao, K., and S. N. Cohen. (2003). Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomerebinding protein essential for linear DNA replication. Genes Dev. 17: 774–785.

3、 Bao, K., and S. N. Cohen. (2004). Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex. Proc. Natl. Acad. Sci. USA 101:14361–14366.

51: 1615–1628.

5、 Birch, A., Häusler, A., and Hütter, R.(1990). Genome rearrangement and genetic instability in Streptomyces spp. Journal of bacteriology. 172, 4138-4142.