耐熱性蛋白Sac7d多聚體設計結構之決定

Abstract

Sac7d蛋白質是一個在耐熱性古生菌Sulfolobus acidocaldarius發現的小分子帶鹼性的染色體蛋白，Sac7d對於溫度、酸性環境以及化學物質有很高的耐受性，它對DNA序列並沒有特定的專一性，一般認為它的功能是維持Sulfolobus acidocaldarius的染色質在高溫環境中的穩定性。Sac7d是以單體型式存在，我們認為多聚體的Sac7d 應該會表現出與單體Sac7d不同的DNA結合性質。為了得到多聚體的Sac7d，利用蛋白質工程的方法將一段a螺旋的肚肚接在Sac7d的尾端，理論上藉著螺旋—螺旋作用力形成的leucine zipper，兩個或更多的單體Sac7d會因此而結合成一個多聚體。先在電腦模擬下確認此方法的可行性，利用大腸桿菌大量表現此重組蛋白，根據其性質，在加熱後去除大部分的雜蛋白，再藉由陽離子交換管柱層析法純化，即可得到純度95％以上的產物。藉由分子篩管柱層析、化學鏈結實驗、表面物質共振實驗與圓二色光譜分析探討其物理及化學特性。以分子篩管柱層析、化學鏈結實驗、表面物質共振實驗證明水溶液下多聚體存在的事實，圓二色光譜實驗確認此融合蛋白二級結構的組成及其對雙股DNA構型的影響，利用表面物質共振實驗得到其對雙股DNA的動力學數據，從x光晶體燒射實驗得到蛋白質立體結構。此次的設計可以當作一個成功的範例—如何賦予蛋白質額外的功能卻又不會使原來的性質喪失，更進一步的，也許能以此方式得到一個衍生自Sac7d的耐熱性DNA酵素，如同Tag DNA聚合?應用於PCR上，或是甚至於外加上一段穿透細胞膜模組，可以如同膜蛋白一般鑲嵌在微胞上，當成一個高穩定性的DNA載體，應用於基因治療上。

The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Sac7d is extremely stable to heat, acid and chemical agents. This protein binds DNA without any particular sequence preference. We propose that multimeric Sac7d would show different DNA binding properties. In order to form dimeric Sac7d, a helical peptide was fused after the C-terminal end of Sac7d. Two or more monomers theoretically will interact with each other via hydrophobic force, as in leucine zipper, to form a dimer. Large amount of recombinant Sac7d was obtained by expression in E. Coli and purification by heating and ion-exchange chromatography. The physical and chemical properties of Sac7d were verified by gel-filtration, chemical cross-linking with SDS-PAGE, SPR, and CD. The tertiary structure was determined by X-ray crystallography. This model works as a successful template that endows protein new function without losing original properties. Furthermore, a thermo-stable enzyme that targets DNA, such as Tag DNA polymerase using in PCR, deriving from Sac7d might be practicable, or fusing a transmembrane domain to Sac7d which inlays in micelles works as a highly stable DNA carrier used in gene therapy.