Protein phosphatase 1(PP1) 是一個主要的絲胺酸/蘇胺酸蛋白質去磷酸酶. Hepp1 (heart-specific protein phosphatase1-binding protein)是一個我們實驗室找到的PP1 結合蛋白質. HePP1與PP1 結合後並不影響其酵素活性. 這個特殊的交互作用有必要進一步探討其特性. 在此研究, 我們設計一個新而有效的方法製備重組(recombinant) HePP1蛋白質. 純化過的HePP1未來會被使用在各種結構與化學特性的研究上. 原先使用的質體 pET32a-Trx-Ub-HePP1, 經由DNA定序, 發現帶有兩個點變異在HePP1的序列上. 我們設計了連續兩次的點突變實驗(site-directed mutagenesis)將這兩個點突變校正回來. 接著我們使用Luria-Bertani (LB) broth培養含有校正好質體的E. coli BL21(DE3), 以IPTG 誘導大量的重組Trx-Ub-Hepp1蛋白製造出來. Trx-Ub-Hepp1 融合蛋白經過Ni2+-Sepharose chromatography 然後 SP-Sepharose chromatography 的連續純化. 含有融合蛋白的部分收集起來, 再對蒸餾水進行透析去除各種試劑, 得到相對純的蛋白質. 再經由Yeast ubiquitin C-terminal hydrolase 1(YUH-1) 將 HePP1 切割出來. 最後再以SP-Sepharose chromatography 分離出Hepp1, 經過凍乾後加以儲存. 我們也利用此純化方法製備13C,15N標定的Hepp1.

Protein phosphatase 1 is one of the major serine/threonine protein phosphatases. Hepp1 (heart-specific protein phosphatase1-binding protein) is one of our identified PP1-regulatory proteins. Hepp1 binds to PP1, but does not inhibit enzyme activity. This special interaction between Hepp1 and PP1 needs to be characterized. In this study, I designed the new and efficient methods to prepare the recombinant Hepp1. The purified Hepp1 will be used for the structural and biochemical studies in the future. The original plasmid, pET32a-Trx-Ub-Hepp1, carried two mutation sites in the Hepp1 region after analysis by DNA sequencing. A two-step site-directed mutagenesis was performed to correct the mutations. DNA sequencing analysis demonstrated that both mutations of Hepp1 have been corrected. E. coli BL21(DE3) harboring with the corrected vector, pET32a-Trx-Ub-Hepp1, was grown in LB broth and the recombinant Trx-Ub-Hepp1 was induced to expression by addition of IPTG. Trx-Ub-Hepp1 fusion protein was purified by Ni2+-Sepharose chromatography and then followed by SP-Sepharose chromatography. Fractions containing Trx-Ub-Hepp1 were pooled and dialyzed against distilled water. Hepp1 was cleaved from Trx-Ub-Hepp1 by YUH-1. The released Hepp1 was further purified by SP-Sepharose chromatography. Fractions containing Hepp1 were pooled, lyophilized, and stored. I also used the purification methods to prepare 13C, 15N-labeled Hepp1.

中文摘要 ii
英文摘要(Abstract) iii
目錄 1
圖目錄 2
Introduction 3
Rationale 6
Materials and Methods 7
Site-directed mutagenesis 7
Examine the expression of Trx-Ub-Hepp1 induced by IPTG 8
Purification of the recombinant Hepp1 9
Preparation of 13C,15N-labeled Hepp1 11
Purification of yeast ubiquitin C-terminal hydrolase-1 (YUH1) 12
Results and Discussion 13
Construction of Hepp1 expression vector 13
Purification of the recombinant Hepp1 14
Preparation of the 13C,15N-labeled Hepp1 16
文獻 18
Figures and Legends 19
附錄 29

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