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研究生:黃智源
研究生(外文):Chih Yuan Huang
論文名稱:HePP1之製備
論文名稱(外文):Preparation of the Recombinant HePP1
指導教授:黃憲斌
指導教授(外文):Hsien-bin Huang
口試委員:李沁林達顯楊肇基
口試委員(外文):Chin LiTa-Hsien LinYang, J. J
口試日期:2014-07-25
學位類別:碩士
校院名稱:國立中正大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:30
中文關鍵詞:PP1HePP1
外文關鍵詞:PP1HePP1
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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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2. Cohen PTW: Protein phosphatase 1 – targeted in many directions. Journal of Cell Science, 115: 241-256, 2002
3. Egloff MP, Johnson DF, Moorhead G, Cohen PTW, Cohen P, Barford D: Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1, 1997
4. Lin T-H, Tsai P-C, Liu H-T, Chen Y-C, Wang L-H, Hsieh F-K, Huang H-B: Characterization of the Protein Phosphatase 1–Binding Motifs of Inhibitor-2 and DARPP-32 by Surface Plasmon Resonance. Journal of Biochemistry, 138: 697-700, 2005
5. Huang H-b, Horiuchi A, Watanabe T, Shih S-R, Tsay H-J, Li H-C, Greengard P, Nairn AC: Characterization of the Inhibition of Protein Phosphatase-1 by DARPP-32 and Inhibitor-2. Journal of Biological Chemistry, 274: 7870-7878, 1999
6. Goldberg J, Huang H-b, Kwon Y-g, Greengard P, Nairn AC, Kuriyan J: Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature, 376: 745-753, 1995
7. Hurley TD, Yang J, Zhang L, Goodwin KD, Zou Q, Cortese M, Dunker AK, DePaoli-Roach AA: Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2. Journal of Biological Chemistry, 282: 28874-28883, 2007
8. Godet AN, Guergnon J, Maire V, Croset A, Garcia A: The Combinatorial PP1-Binding Consensus Motif (R/K)x (0,1)V/IxFxx(R/K)x(R/K) Is a New Apoptotic Signature. PLoS ONE, 5: e9981, 2010
9. Chen C-Y, Lai N-S, Yang J-J, Huang H-l, Hung W-C, Li C, Lin T-H, Huang H-b: FLJ23654 encodes a heart protein phosphatase 1-binding protein (Hepp1). Biochemical and Biophysical Research Communications, 391: 698-702, 2010
10. Kao S-C, Chen C-Y, Wang S-L, Yang J-J, Hung W-C, Chen Y-C, Lai N-S, Liu H-T, Huang H-l, Chen H-C, Lin T-H, Huang H-b: Identification of phostensin, a PP1 F-actin cytoskeleton targeting subunit. Biochemical and Biophysical Research Communications, 356: 594-598, 2007

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