純化與表面分析鏈黴菌端點蛋白

鏈黴菌(streptomyces)的染色體是線性的，有些也含有線性質體，它們的5’端有一個以共價鍵連接的蛋白稱為端點蛋白(TP)。而其複製是由中間向兩端，如此會造成末端無法補足的問題，而端點蛋白在末端補足的機制中可能扮演引子的角色。端點蛋白(TpgC)由185個胺基酸組成，實驗結果推測第114位置的蘇胺酸與染色體末端產生共價鍵結。純化已將鏈黴菌端點蛋白TpgC上114蘇胺酸突變成胱胺酸的 TpgC114C蛋白並且研究端點蛋白的表面結構。本論文主要將tpgC114C 選殖到pET22-b表現載體然後轉殖到大腸桿菌中表現，利用IPTG去誘發蛋白的產生，再以鎳離子親合管純化C端含有His-tag的TpgC114C蛋白，得到純度80 % 濃度約0.2 mg/ml的蛋白。另外﹐蛋白表面研究 : 第一、利用固定化金屬親合力層析法(immobilized metal affinity chromatography，IMAC)來探針蛋白表面組胺酸殘基，本論文選用銅離子來偵測TpgC蛋白序列中的三個組胺酸，結果發現TpgC蛋白沒有組胺酸殘基存在於蛋白表面。第二、利用蛋白水解酶LysC進行partial digestion，產生17kDa與12kDa水解片段，利用西方墨漬法去證實端點蛋白表面切點可能為K 57 和K 204。

關鍵字

端點蛋白；鏈黴菌；純化

並列摘要

Streptomyces species have linear chromosomes and linear plasmids. Both of them have a 5’ covalent-bound protein called terminal protein (TP). Bidirectional replication happens in the center of chromosome. It may make the terminal protein as primer to complete the end replication. TpgC consists of 185 amino acids. Through some experiments, it was suggested that the 114th threonine might be covalently bound to the chromosome end. TpgC114C, which was mutated at the 114th threonine to cystein, was purified and protein surface was analyzed. tpgC114C was cloned into pET22-b expression vector and transformed into E.coli. Upon induction with IPTG, the recombinant protein with a C-terminal His-tag was purified by Ni2＋resin affinity chromatography. To investigate the protein surface, we probed the surface topography of histidine by Cu2＋resin affinity chromatography. TpgC contains three histidines, none of them was on the surface according to our IMAC results. Also, partial digestion of TpgC114C by LysC was performed to generate fragments of 17kDa and 12kDa. We proposed that they resulted from the cleavages at K57 and K204 by western examination.

並列關鍵字

Purification ； Terminal protein ； Streptomyces

參考文獻

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純化與表面分析鏈黴菌端點蛋白

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