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研究生:王厚仁
研究生(外文):Hou Ren Wang
論文名稱:人類(月太)基甘胺酸-阿爾法-醯胺化單加氧酵素的功能性表現
論文名稱(外文):Functional Expression of Human Peptidylglycine alpha-Amidating Monooxygenase
指導教授:王成德胡帥讓
指導教授(外文):C. T. WangDaniel Hu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:英文
論文頁數:69
中文關鍵詞:醯胺化S2 細胞株(月太)基甘胺酸-阿爾法-醯胺化單加氧酵素
外文關鍵詞:amidationS2 cellpeptidylglycine alpha-amidating monooxygenase
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中文摘要
許多肽類荷爾蒙其C端必須被醯胺化,才能表現其生物活性。 在較高等的生物中,肽類荷爾蒙的前驅物-肽基甘胺酸的醯胺化作用為一兩步驟的反應,由一具雙重功能的酵素所催化- 肽基甘胺酸-阿爾法-醯胺化單加氧酵素,簡稱PAM. 在基因重組肽類藥物的生產上,PAM扮演了重要的角色,然而,PAM酵素目前尚未商品化。 雖然肽類的醯胺化作用也可利用化學法來達成,然而其存在著許多缺點令酵素催化法為一較佳的選擇。 在本研究中,嘗試了數種蛋白質表現系統,以發展出一套更經濟的方式,來生產PAM。 首先,將人類的PAM基因轉染到中國倉鼠卵巢細胞株-K1進行表現,轉錄由巨細胞病毒啟動子所調控。 利用西方墨點轉印技術,可在細胞培養基中偵測一個95千道耳吞的蛋白質,與PAM的分子量近似。 接著我們進一步嘗試酵母菌表現系統,將PAM基因與酵母菌阿爾法配對因子的領導序列融合,或利用PAM本身的訊息序列,使PAM在酵母菌做分泌式的表現,轉錄由gal1啟動子調控。 然而,我們並未偵測到PAM蛋白質或其活性。 若將訊息序列去除,改為在酵母菌胞內表現,結果表現出的PAM卻是不可溶並且無活性的。 於是我們又嘗試用大腸桿菌的T7表現系統,在大腸桿菌胞內表現PAM與Intein的融合蛋白,結果也是形成無活性的包涵體。 最後,利用昆蟲細胞表現系統,可生產出具有功能的PAM酵素,轉錄由metalothionein啟動子調控。 產量可達5毫克/公升。

Abstract
Many peptide hormones must be amidated at their carboxyl termini for full biological activity. In higher organisms, the enzymatic formation of C-terminally amidated peptides is a two-step process catalyzed by a single bifunctional enzyme, peptidylglycine alpha amidating monooxygenase (PAM). PAM is important in production of recombinant pharmaceutical peptides that require amidation. Although there are some chemical methods for peptide alpha amidation, several existing drawbacks make the enzymatic way the favorite one. However, recombinant PAM enzyme was not available commercially. Thus, we have applied different expression systems in this study to produce economical PAM for industrial use. The commercial human PAM (hPAM) cDNA was initially expressed in Chinese hamster ovary cell line (CHO-K1) to confirm the correct expression of desired proteins. A 98 kD protein band of expected size was detected by Western blotting. To provide a rapid and cost-effective expression of hPAM, a secretory system by using the leader sequence of yeast alpha mating factor was primary selected. However, neither PAM protein nor its activity was detected through this approach. Further, by deleting the signal sequence, cytosolic expression of hPAM in yeast resulted in insoluble and function-less enzyme. Finally, secretory expression of the truncated version of the bi-functional PAM (31aa-817aa) by fusing with the Bip signal sequence was performed in the Drosophila Schneider 2 (S2) cell line. Under the regulation of metallothionein promoter, 5 g/ml of hPAM31-817 was obtained with continuous induction of 10 M CdCl2 for five days and a simple metal chelate resin purification procedure.

Table of contents
Acknowledgement(Chinese)…………………………iii
List of Tables and Figures………………………iv
Abbreviations………………………………………..v
Abstract (Chinese)……………………………..…vii
Abstract (English) …………………………………1
Introduction……………………………………………2
Materials and methods………………………………9
Results…………………………………………………29
Discussion……………………………………………52
References……………………………………………56
Appendices……………………………………………61

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