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研究生:羅鈺惠
研究生(外文):Yu-Hui Lo
論文名稱:含RGD序列蛋白的表現及特性之研究,ADAM15與γ-雨傘節毒素
論文名稱(外文):Expression and Characterization of RGD-Containing Proteins, a Disintegrin and Metalloprotease 15 (ADAM 15) and γ-Bungarotoxin
指導教授:莊偉哲
指導教授(外文):Woei-Jer Chuang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:107
中文關鍵詞:酵母菌系統核磁共振儀γ-雨傘節毒素亞當蛋白15
外文關鍵詞:Pichia pastorisADAM15γ-BungarotoxinNMR
相關次數:
  • 被引用被引用:1
  • 點閱點閱:274
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  • 下載下載:46
  • 收藏至我的研究室書目清單書目收藏:1
ADAM 15 (A Disintegrin And Metalloprotease 15) 又稱為metargidin (metalloprotease -RGD-disintegrin protein) 是一種型態I跨膜性醣蛋白 (type I transmembrane glycol- protein)。ADAM15 共含 814 個胺基酸,根據序列分析,ADAM15 共有前區域 (pro domain)、金屬蛋白區 (metalloprotease domain)、類去組合蛋白區 (disintegrin-like domain)、多半胱胺酸區 (cysteine-rich domain)、類上皮細胞生長因子區 (EGF-like domain)、跨膜區 (transmembrane domain) 以及細胞質區 (cytoplasmic domain) 等七個區域。ADAM15 的去組合蛋白區域是 ADAM 家族中唯一在去組合蛋白區中有 RGD 序列。文獻中指出 ADAM15 可利用去組合蛋白區上的RGDCD (胺基酸484至488) 序列與組合蛋白 (integrin) avb3、a5b1 發生交互作用。而既然 ADAM15 的去組合蛋白區可以當作組合蛋白中的 avb3 與 a5b1 的受體,因此它可能在發育與病理時期的細胞與細胞之間的交互作用上具有決定性的影響。有趣的是,含 91 個胺基酸 (胺基酸 420 至 510) 的去組合蛋白區中有 15 個半胱胺酸 (cysteine);含 146 個胺基酸(胺基酸 511 至 656) 的多半胱胺酸區中有 13 個半胱胺酸以及兩個潛在的 N 連結醣化位置。去組合蛋白區與多半胱胺酸區 (胺基酸 420 至 656) 共含有 28 個半胱胺酸。
本實驗室假設多半胱胺酸區可能牽涉到與去組合蛋白區的雙硫鍵形成。而為瞭解ADAM15 去組合蛋白區與多半胱胺酸區的功能上結構關係,本實驗室決定大量表現並純化這些區域。我們從 Takada教授實驗室取得構築在大腸桿菌系統 pGEX-2T 上 ADAM15的去組合蛋白區之構築體。但是由大腸桿菌系統表現的蛋白並不適合作為多雙硫鍵蛋白之研究,因此本實驗室選擇將設計有His-tag的 ADAM15s (s表示secreted protein) 構築於pPICZaB載體上,以酵母菌系統 (Pichia pastoris) 表現,接著以鎳離子螯合親和性色層分析法、P-10管柱以及高效能液相層析分析法純化。根據實驗結果,發現此蛋白質會利用 RGDCD 序列中的半胱胺酸形成分子間的雙硫鍵,而產生二聚體,這證明 RGDCD 中的半胱胺酸可能與 ADAM15 中其他的半胱胺酸鍵結形成分子內雙硫鍵。為了避免二聚體的產生,首先我們將此半胱胺酸以定點突變的方式構築出C487S突變株,亦以酵母菌系統表現及純化,然後以用血小板凝集試驗測定其抑制血小板凝集的能力,當濃度高達50 mM時,仍沒有抑制血小板凝集的現象。以核磁共振與旋光光譜分析發現所純化到的C487S突變蛋白的折疊並不正確。接著我們構築出C端多五個胺基酸的ADAM15-96s,用以探討RGDCD序列的C487會與 ADAM15中的哪一個半胱胺酸鍵結。而根據質譜儀的結果,我們得到單體的ADAM15-96s,這證實本實驗室假設RGDCD序列能與ADAM15中其他的半胱胺酸鍵結而形成分子內雙硫鍵。但是以NMR研究,發現此蛋白質的結構並不正確。因此我們試著設計帶有His-tag的ADAM15-96i (i表示intracellular protein) 構築於pPICZC載體上,接著構築ADAM15-111s與ADAM15-127s於酵母菌系統,目前仍在進行純化ADAM15蛋白,本實驗室未來將繼續完成構築ADAM15去組合蛋白區與多半胱胺酸區於酵母菌系統和表現、純化、功能分析,以確定ADAM15中這兩個區域的功能與結構之關係。
另外本實驗室研究另一個含有 RGDGP 序列的蛋白:γ-Bungarotoxin。它最早是由Bungarus multicinctus 中純化而來,屬於一種蛇毒蛋白。它全長共有 68 個胺基酸,包括10 個多半胱胺酸 (cysteine residue) 以及在 33 至 37 位置上有一段 RGDGP 序列。根據本實驗室以 NMR 研究此蛋白質的結果,目前已經完成 γ-Bungarotoxin 的 3D 結構,並認為 γ-Bungarotoxin 的 3D 結構包含兩股與三股的 b-sheets (the double- and triple-stranded antiparallel b-sheets) 以及三個 loops. 其中位在 loop II 上的 RGD 序列能與 integrin 結合而形成一個 reverse turn 的結構,這結構與 disintegrin 的活化區類似。然而本實驗室秋月學姐將此蛋白做血小板凝集測試發現 γ-Bungarotoxin 的 IC50 值為 34 mM,比其它 disintegrin 減少 200 倍。因此我們構築 ADAM 15 (A Disintegrin And Metalloprotease 15) 又稱為metargidin (metalloprotease -RGD-disintegrin protein) 是一種型態I跨膜性醣蛋白 (type I transmembrane glycol- protein)。ADAM15 共含 814 個胺基酸,根據序列分析,ADAM15 共有前區域 (pro domain)、金屬蛋白區 (metalloprotease domain)、類去組合蛋白區 (disintegrin-like domain)、多半胱胺酸區 (cysteine-rich domain)、類上皮細胞生長因子區 (EGF-like domain)、跨膜區 (transmembrane domain) 以及細胞質區 (cytoplasmic domain) 等七個區域。ADAM15 的去組合蛋白區域是 ADAM 家族中唯一在去組合蛋白區中有 RGD 序列。文獻中指出 ADAM15 可利用去組合蛋白區上的RGDCD (胺基酸484至488) 序列與組合蛋白 (integrin) avb3、a5b1 發生交互作用。而既然 ADAM15 的去組合蛋白區可以當作組合蛋白中的 avb3 與 a5b1 的受體,因此它可能在發育與病理時期的細胞與細胞之間的交互作用上具有決定性的影響。有趣的是,含 91 個胺基酸 (胺基酸 420 至 510) 的去組合蛋白區中有 15 個半胱胺酸 (cysteine);含 146 個胺基酸(胺基酸 511 至 656) 的多半胱胺酸區中有 13 個半胱胺酸以及兩個潛在的 N 連結醣化位置。去組合蛋白區與多半胱胺酸區 (胺基酸 420 至 656) 共含有 28 個半胱胺酸。
本實驗室假設多半胱胺酸區可能牽涉到與去組合蛋白區的雙硫鍵形成。而為瞭解ADAM15 去組合蛋白區與多半胱胺酸區的功能上結構關係,本實驗室決定大量表現並純化這些區域。我們從 Takada教授實驗室取得構築在大腸桿菌系統 pGEX-2T 上 ADAM15的去組合蛋白區之構築體。但是由大腸桿菌系統表現的蛋白並不適合作為多雙硫鍵蛋白之研究,因此本實驗室選擇將設計有His-tag的 ADAM15s (s表示secreted protein) 構築於pPICZaB載體上,以酵母菌系統 (Pichia pastoris) 表現,接著以鎳離子螯合親和性色層分析法、P-10管柱以及高效能液相層析分析法純化。根據實驗結果,發現此蛋白質會利用 RGDCD 序列中的半胱胺酸形成分子間的雙硫鍵,而產生二聚體,這證明 RGDCD 中的半胱胺酸可能與 ADAM15 中其他的半胱胺酸鍵結形成分子內雙硫鍵。為了避免二聚體的產生,首先我們將此半胱胺酸以定點突變的方式構築出C487S突變株,亦以酵母菌系統表現及純化,然後以用血小板凝集試驗測定其抑制血小板凝集的能力,當濃度高達50 mM時,仍沒有抑制血小板凝集的現象。以核磁共振與旋光光譜分析發現所純化到的C487S突變蛋白的折疊並不正確。接著我們構築出C端多五個胺基酸的ADAM15-96s,用以探討RGDCD序列的C487會與 ADAM15中的哪一個半胱胺酸鍵結。而根據質譜儀的結果,我們得到單體的ADAM15-96s,這證實本實驗室假設RGDCD序列能與ADAM15中其他的半胱胺酸鍵結而形成分子內雙硫鍵。但是以NMR研究,發現此蛋白質的結構並不正確。因此我們試著設計帶有His-tag的ADAM15-96i (i表示intracellular protein) 構築於pPICZC載體上,接著構築ADAM15-111s與ADAM15-127s於酵母菌系統,目前仍在進行純化ADAM15蛋白,本實驗室未來將繼續完成構築ADAM15去組合蛋白區與多半胱胺酸區於酵母菌系統和表現、純化、功能分析,以確定ADAM15中這兩個區域的功能與結構之關係。
另外本實驗室研究另一個含有 RGDGP 序列的蛋白:γ-Bungarotoxin。它最早是由Bungarus multicinctus 中純化而來,屬於一種蛇毒蛋白。它全長共有 68 個胺基酸,包括10 個多半胱胺酸 (cysteine residue) 以及在 33 至 37 位置上有一段 RGDGP 序列。根據本實驗室以 NMR 研究此蛋白質的結果,目前已經完成 γ-Bungarotoxin 的 3D 結構,並認為 γ-Bungarotoxin 的 3D 結構包含兩股與三股的 b-sheets (the double- and triple-stranded antiparallel b-sheets) 以及三個 loops. 其中位在 loop II 上的 RGD 序列能與 integrin 結合而形成一個 reverse turn 的結構,這結構與 disintegrin 的活化區類似。然而本實驗室秋月學姐將此蛋白做血小板凝集測試發現 γ-Bungarotoxin 的 IC50 值為 34 mM,比其它 disintegrin 減少 200 倍。因此我們構築 γ-Bungarotoxin 於 pPICZaB 載體上 (可分泌至細胞外) 以酵母菌系統 (Pichia pastoris) 表現,並以鎳離子螯合親和性色層分析法以及高效能液相層析分析法純化後,以研究其結構與功能之間的關係。
而根據過去本實驗利用 Pichia pastoris 表現系統來表現蛇毒 rhodostomin (具有六對雙硫鍵) 的研究,結果得到折疊正確結構的蛋白,但是對於ADAM15-96s (具有十六對雙硫鍵)、ADAM15-111s (具有十八對雙硫鍵) 以及 g-Bungarotoxin (具有五對雙硫鍵) 等蛋白的表現,卻是得到結構折疊錯誤的蛋白,因此我們認為 Pichia pastoris 表現系統並不適合表現所有具高度雙硫鍵結的蛋白。γ-Bungarotoxin 於 pPICZaB 載體上 (可分泌至細胞外) 以酵母菌系統 (Pichia pastoris) 表現,並以鎳離子螯合親和性色層分析法以及高效能液相層析分析法純化後,以研究其結構與功能之間的關係。
而根據過去本實驗利用 Pichia pastoris 表現系統來表現蛇毒 rhodostomin (具有六對雙硫鍵) 的研究,結果得到折疊正確結構的蛋白,但是對於ADAM15-96s (具有十六對雙硫鍵)、ADAM15-111s (具有十八對雙硫鍵) 以及 g-Bungarotoxin (具有五對雙硫鍵) 等蛋白的表現,卻是得到結構折疊錯誤的蛋白,因此我們認為 Pichia pastoris 表現系統並不適合表現所有具高度雙硫鍵結的蛋白。
ADAM15 (A Disintegrin And Metalloprotease 15) is also known as metargidin (metalloprotease-RGD-disintegrin protein) which is a multi-domain type I transmembrane glycoprotein consisting of 814 amino acids. ADAM15 is the only known ADAM family that has an RGD motif in the disintegrin domain. It can interact with integrin avb3 and a5b1 via the RGDCD motif of the disintegrin domain. Since the disintegrin domain of ADAM15 serves as a ligand for integrin avb3 and a5b1, it may play crucial roles in cell-cell interaction during development and in pathological conditions. Based on previous report, the disintegrin-like domain is from residue 420 to 510 and contains 15 cysteine residues. The cysteine-rich domain is from residue 511 to 656 and contains 13 cysteine residues. Since these domains have odd cysteine residues, we hypothesize that the cysteine-rich domain may involve in forming disulfide bonds with the disintegrin domain. Therefore, we expressed the ADAM15 from residue 420 to 656 by extensive PCR method. Since proteins expressed in E. coli expression system are not suited for studying highly disulfide-bonded proteins, we expressed the proteins in Pichia pastoris to avoid the misfolding problem. The expressed proteins have been purified to homogeneity by Ni2+-chelating and RP-HPLC chromatography. SDS-PAGE analysis indicated that the purified ADAM15-91s (s means secreted protein) protein exhibits a mixture of monomer and dimmer, suggesting that one of 15 cysteine residues in ADAM15-91s formed an intermolecular disulfide bond. Therefore, we mutated the cysteine 487 to serine and constructed ADAM15-96s, which contains five additional amino acids (PCAGG) in C-terminus to avoid intermolecular disulfide bond. Results from CD and NMR studies indicate that the C487S mutant was misfolded, suggesting that cysteine 487 is involved in proper folding of ADAM15. The cysteine-rich domain of ADAM15 (ADAM15-146) contains 146 amino acids from residue 511 to 656 with 13 cysteines. Therefore, we used the extensive PCR method to construct the disintegrin and cysteine-rich domains of ADAM15 and expressed them in Pichia pastoris.
γ-Bungarotoxin is a snake venom protein isolated from Bungarus multicinctus and contains 68 amino acids, including 10 cysteine residues and an RGDGP sequences at positions of 33-37. In our NMR study, the 3D structure of γ-Bungarotoxin was determined. It contains the double- and triple-stranded antiparallel b-sheets and three loops. The RGD motif, the binding site for integrin, lies at the loop II in a reverse turn conformation which is similar to that of the active site of disintegrins. However, we found that γ-Bungarotoxin inhibits platelet aggregation with an IC50 of 34 mM which is 200-fold decrease compared to other disintegrin. Therefore, we have expressed γ-Bungarotoxin in Pichia pastoris and study on its function and structure relationships are going.
中文摘要……………………………………………………I
英文摘要…………………………………………………III
誌謝………………………………………………………IV
目錄…………………………………………………………V
圖目錄……………………………………………………IX
表目錄……………………………………………………XI
附錄目錄…………………………………………………XII
縮寫檢索表……………………………………………XIII
儀器………………………………………………………XIV
第1章 緒論………………………………………………1
1-1 ADAM15部分…………………………………………1
1-1-1 組合蛋白 (Integrin) 之介紹…………………1
1-1-2 去組合蛋白 (Disintegrin) 之介紹…………3
1-1-3 ADAM家族之介紹…………………………………4
1-1-4 ADAM15之介紹……………………………………6
1-1-5 ADAM15的去組合蛋白區與蛇毒Bitistatin去組合蛋白之比較……8
1-1-6 論文研究動機及內容之簡介……………………9
1-2 gamma-Bungarotoxin部分…………………………11
1-2-1 分泌毒液毒蛇之簡介……………………………11
1-2-2 毒蛇所分泌毒液之簡介…………………………11
1-2-3 神經毒素 (Neurotoxin) 之簡介………………13
1-2-4 雨傘節 (Bungarus multicinctus) 之簡介……13
1-2-5 gamma-Bungarotoxin之簡介……………………14
1-2-6 論文研究動機及內容之簡介…………………14
第2章 材料及方法………………………………………16
2-1 實驗菌株、質体與培養基配方……………………16
2-1-1 Host strains and genotypes…………………16
2-1-2 Vector……………………………………………16
2-1-3 Growth medium…………………………………17
2-2 基因之來源…………………………………………18
2-2-1 ADAM15去組合蛋白區基因之來源………………18
2-2-2 gamma-Bungarotoxin基因之來源………………18
2-3 重組基因之構築……………………………………18
2-3-1 ADAM15去組合蛋白區與多半胱胺酸區
(ADAM15-96i與ADAM15-111s) 重組基因之構築………18
2-3-1-1 聚合酵素連鎖反應 (Polymerase Chain Reaction; PCR)……20
2-3-1-2 構築PCR片段於pPICZaB或pPICZC質體中……20
2-3-1-3 E. coli形質轉移 (transformation)………23
2-3-1-4 用菌的PCR來篩選E. coli 菌株………………24
2-3-1-5 Pichia Pastoris形質轉移……………………25
2-3-1-6 用菌的PCR來篩選Pichia pastoris 菌株……26
2-3-2 gamma-Bungarotoxin基因重組基因之構築……27
2-3-2-1 聚合酵素連鎖反應 (Polymerase Chain Reaction; PCR)……27
2-3-2-2 構築PCR片段於pPICZaA質體中………………28
2-3-2-3 E. coli形質轉移 (transformation)………30
2-3-2-4 用菌的PCR來篩選E. coli 菌株………………31
2-3-2-5 Pichia Pastoris形質轉移 …………………32
2-3-2-6 用菌的PCR來篩選Pichia pastoris菌株……34
2-4 重組蛋白的表現及純化……………………………34
2-4-1 ADAM15去組合蛋白區與多半胱胺酸區
(ADAM15-96i、ADAM15-96s與ADAM15-111s) 重組蛋白的表現及純化…34
2-4-1-1 重組蛋白的表現………………………………34
2-4-1-2 重組蛋白的純化………………………………35
2-4-1-2-1 ADAM15-96i重組蛋白的純化………………35
2-4-1-2-2 ADAM15-96s重組蛋白的純化………………37
2-4-1-2-3 ADAM15-111s重組蛋白的純化………………39
2-4-2 gamma-Bungarotoxin重組蛋白的表現及純化…41
2-4-2-1 重組蛋白的表現………………………………41
2-4-2-2 重組蛋白的純化………………………………42
2-5 SDS-PAGE 分析……………………………………43
2-6質譜 (Mass) 之分析…………………………………46
2-7血小板凝集之分析……………………………………46
2-8核磁共振 (Nuclear Magnetic Resonance,NMR) 之分析………47
第3章 結果………………………………………………49
3-1 重組基因的製備……………………………………49
3-1-1 ADAM15去組合蛋白與多半胱胺酸區
(ADAM15-96i與ADAM15-111s) 部分……………………49
3-1-2 gamma-Bungarotoxin部分………………………50
3-2 重組蛋白的製備……………………………………51
3-2-1 ADAM15去組合蛋白與多半胱胺酸區
(ADAM15-96i、ADAM15-96s與ADAM15-111s) 部分……51
3-2-2 gamma-Bungarotoxin部分………………………51
3-3 重組蛋白之SDS-PAGE分析…………………………52
3-3-1 ADAM15去組合蛋白與多半胱胺酸區
(ADAM15-96s與ADAM15-111s) 部分…………………52
3-3-2 gamma-Bungarotoxin部分………………………52
3-4 重組蛋白之功能分析----血小板凝集之分析……52
3-4-1 ADAM15去組合蛋白與多半胱胺酸區
(ADAM15-96s與ADAM15-111s) 部分…………………52
3-4-2 gamma-Bungarotoxin部分………………………53
3-5 重組蛋白之功能分析----質譜分析………………53
3-5-1 ADAM15去組合蛋白與多半胱胺酸區
(ADAM15-96s與ADAM15-111s) 部分……………………53
3-5-2 gamma-Bungarotoxin部分………………………54
3-6 重組蛋白之功能分析----核磁共振儀分析結構…54
3-6-1 ADAM15去組合蛋白與多半胱胺酸區
(ADAM15-96i、ADAM15-96s與ADAM15-111s) 部分……54
3-6-2 gamma-Bungarotoxin部分………………………55
第4章 結論…………………………………………56
4-1 ADAM15部分……………………………………56
4-2 gamma-Bungarotoxin部分……………………58
第5章 討論…………………………………………59
參考文獻……………………………………………61
圖……………………………………………………69
表……………………………………………………95
附錄……………………………………………………99
自述…………………………………………………107
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