臺灣博碩士論文加值系統

人類胱硫醚乙型合成酶（hCBS）為含硫胺基酸代謝中的關鍵酵素，透過轉硫作用的進行，可以代謝有毒的中間產物，高半胱胺酸，再進一步生合成人體所需的胺基酸，半胱胺酸（cysteine）；除此之外，hCBS也參與麩胱甘肽以及H2S的生成。近期研究發現，S-腺苷甲硫胺酸（S-adenosylmethionine，SAM）與hCBS結合後，會引起hCBS構型的改變並且調節酵素本身的活性，而這一項研究也揭示了蛋白質構型特性與結合小分子之間微妙的關係。已知目前有許多小分子會與hCBS結合，不過，小分子對hCBS折疊性質的影響仍然未知，因此，我們將進一步探討小分子對hCBS構型折疊的影響。在本研究中，我們成功建構了兩種含有hCBS基因的重組質體並且優化了此蛋白質的純化流程以增加產量，由於產量及純度的考量，我們最後選擇hCBS-pET32a作為蛋白質的表現質體用於純化製程。我們利用Pulse proteolysis進行hCBS的熱力學穩定度實驗，實驗結果發現，hCBS可能是一個具有多重構型狀態的蛋白質，而SAM以及cysteine會針對特定構型產生影響；另外，我們也進行了hCBS的展開動力學實驗，結果顯示了，SAM以及cysteine對於hCBS的展開速度有著不同的影響。而這樣的結果提供我們有關於小分子參與代謝調控的參考資訊，也許可以進一步應用在疾病治療以及藥物的開發相關研究上。

Human cystathionine β-synthase（hCBS）is a key enzyme in sulfur amino acid metabolism. This enzyme can catabolize of homocysteine, which is a toxic metabolite, via transsulfuration. Moreover, hCBS also participates in the biosynthesis of cysteiene, glutathionine and H2S. Recently, hCBS was found to be allosterically activated by S-adenosylmethionine, which could trigger a conformational change of hCBS through binding. It reveals that the folding properties of this protein may be potentially affected by ligands binding. Interestingly, there are lots of ligands that interact with hCBS with unclear effect on the folding properties of this enzyme.
In this study, we carefully investigated the ligand binding effect on the folding of hCBS. For obtaining hCBS, we have successfully constructed two recombinant plasmids and optimized the purification conditions for the protein purification. Because of the better purity and production, we chose hCBS-pET32a to express hCBS for subsequent experiments. We have meaured the thermodynamic stability and unfolding kinetics of hCBS by pulse proteolysis. Our results suggest that hCBS is a protein with multiple conformational state and ligands may affect specific conformation. Additionally, SAM and cysteine were found to have different influence on the unfolding rate of hCBS. This study provides valuable information for understanding the role of ligands in the metabolic regulation. This information would be possibly applied to disease treatments and drug development in the future.

摘要...i
Abstract...ii
目次...iii
圖目次...vi
表目次...viii
第一章、 前言...1
一、 甲硫胺酸代謝的重要性...1
(一) 高半胱胺酸代謝...1
(二) 轉硫路徑...1
二、 Cystathionine beta-synthase之生理功能...2
(一) hCBS參與之化學反應...2
(二) Homocystinuria為遺傳性缺乏hCBS之疾病...2
三、 Cystathionine beta-synthase之結構特性...3
(一) hCBS為組合式蛋白質...3
(二) hCBS結構域之間相互調節作用...3
四、 功能性蛋白質與小分子之間之關係...4
(一) 功能性蛋白質之重要性...4
(二) 結合小分子調控功能性蛋白質之重要性...4
第二章、 實驗目的...6
第三章、 實驗架構...7
第四章、 材料與方法...8
一、 hCBS之蛋白質表現及純化...8
(一) 純化pET21a質體...8
(二) 聚合酶連鎖反應放大CBS基因...8
(三) 基因接入質體...9
(四) E.coli BL21（DE3）中大量表現hCBS...11
(五) hCBS純化...11
(六) 聚丙烯醯胺膠體電泳（SDS-PAGE）...14
二、 hCBS之蛋白質表現及純化...16
(一) 純化pET32a質體...16
(二) 聚合酶連鎖反應放大hCBS基因...16
(三) 基因接入質體...16
(四) 大腸桿菌BL21(DE3)中大量表現hCBS...17
(五) hCBS純化...17
三、 西方墨點法（Western Blot）...20
(一) 聚丙烯醯胺膠體電泳（SDS-PAGE）...20
(二) 轉漬 ( Transfer )...20
(三) 阻攔（Blocking）...21
(四) 一級抗體之培養（Primary antibody incubation）... 21
(五) 二級抗體之培養（Secondary antibody incubation）... 21
(六) 檢測（Detection）...21
四、 蛋白質折疊熱力學...22
(一) Pulse Proteolysis...22
五、 蛋白質折疊動力學...23
(一) 展開動力學（unfolding kinetics）...23
第五章、 結果與討論...25
一、 蛋白質表現與純化（hCBS-pET21a）...25
(一) 基因選殖...25
(二) 目標蛋白表現及純化...27
(三) 小結...29
二、 西方墨點法...30
(一) hCBS-pET21a...30
(二) 小結...30
三、 蛋白質表現與純化（hCBS-pET32a）...30
(一) 基因選殖...31
(二) 目標蛋白表現及純化...32
(三) 小結...35
四、 西方墨點法...36
(一) hCBS-pET32a...36
(二) 小結...36
五、 蛋白質折疊熱力學...36
(一) 利用Pulse proteolysis測定unfolding equilibrium... 36
(二) 討論...37
(三) 小結...38
六、 蛋白質折疊動力學...39
(一) 展開動力學（unfolding kinetics）結果...39
(二) 討論...40
(三) 小結...40
第六章、 結論...41
第七章、 參考文獻...42
附錄一、藥品...87
附錄二、套組...88
附錄三、儀器名稱...89
附錄四、縮寫檢索表...90

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