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系統識別號 U0026-0812200915271098
論文名稱(中文) 表現於嗜甲醇酵母菌的馬來亞腹蛇突變蛋白之發酵槽量產最佳化與功能探討
論文名稱(英文) Optimization of the fermentation production in Pichia pastoris and the function of rhodostomin mutants
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 呂宜學
研究生(英文) Yi-Hsueh Lu
學號 s1696101
學位類別 碩士
語文別 中文
論文頁數 110頁
口試委員 指導教授-莊偉哲
口試委員-鄭宏祺
口試委員-周三和
中文關鍵字 去整合蛋白  發酵槽  嗜甲醇酵母菌 
英文關鍵字 fermentation  Pichia pastoris  disintegrin 
學科別分類
中文摘要 整合蛋白是一群位於細胞表現的異質雙體,調整許多細胞步驟,如生長、死亡、黏著、移動。馬來亞腹蛇蛋白為一種有淺力的血小板凝集抑制劑,且屬於去整合蛋白家族一元。馬來亞腹蛇蛋白由68個胺基酸,其序列上帶有48PRGDM53P及6對雙硫所建構而成。我們實驗室先前由就已經成功利用嗜甲醇酵母菌表現馬來亞腹蛇蛋白,且利用它設計出對整合蛋白αvβ3專一性的藥物。本篇研究內容,我主要是做最佳化嗜甲醇酵母菌發酵槽條件,增加嗜甲醇酵母菌表現馬來亞腹蛇突變蛋白質HAS-ARLDDL之產量。比較搖瓶及發酵槽生產馬來亞腹蛇突變蛋白上清液中總蛋白含量,可以得知利用搖瓶產量約為 100 mg/L,而藉由最佳化pH值、溫度、培養基成分、和發酵槽中氧氣供給程度,能使發酵槽上清液總蛋白產量達到 900 mg/L,然而分別純化後產量分別為 30 mg/L 及 170 mg/L。檢視搖瓶及發酵槽兩者純化過程中的回收率分別為30%及17%,似乎是由於發酵槽有偏高的錯誤摺疊蛋白從發酵過程中產生出來。在先前研究我們發現馬來亞腹蛇突變蛋白其連結區域帶為39KKKRTICR47I序列會影響其結合能力及選擇在整合蛋白α5β1, αvβ3, and αIIbβ3,本篇研究中,我已經成功表現三株在連結區域為39KKKRT並含R46A、I47R、R46A/I47R之馬來亞腹蛇突變蛋白及一株在連結區域為39SRAGK並含R46A之馬來亞腹蛇突變蛋白,藉由分析血小板凝集及細胞黏著實驗,結果顯示在連結區域為39SRAGK並含R46A之馬來亞腹蛇突變蛋白會對血小板凝集及整合蛋白αIIbβ3、αvβ3、及 α5β1的活性,分別為 5.4、7.6、2.2、及5.6倍下降。此外連結區域序列為39KKKRT並含R46A之馬來亞腹蛇突變蛋白也會對血小板凝集及整合蛋白αIIbβ3、αvβ3、及 α5β1的活性,分別為 4.1, 24.8, 1.8,及1.8倍下降。而含序列39KKKRT並含 I47R之馬來亞腹蛇突變蛋白能增加對整合蛋白αvβ3、及 α5β1的活性,分別為3、7倍上升。有趣的是,我們發現突變Rho蛋白含39KKKRTIC46AR-ARGDN53P-67Y68H序列對抑制整合蛋白αIIbβ3、αvβ3、及 α5β1之IC50值分別為956.0 nM、5.7 nM、6.6 nM。這項發現可以去除Rho蛋白對整合蛋白αIIbβ3之抑制能力。總體而言,這些突變Rho蛋白之功能研究結果能夠提供有用資訊設計對整合蛋白專一的去整合蛋白,而最佳化表現HAS-ARLDDL蛋白之嗜甲醇酵母菌發酵槽條件,能夠提供一些基本資訊用來表現對整合蛋白專一的去整合蛋白
英文摘要 Integrins are a family of heterodimeric receptors and modulate many cellular processes, including growth, death, adhesion, and migration. Rhodostomin (Rho) is a potent platelet aggregation inhibitor and belongs to the family of disintegrins. Rho consists of 68 amino acids with a 48PRGDM53P motif and six disulfide bonds. In previous study we expressed Rho in Pichia pastrois (P. pastoris) and used it as the scaffold to design integrin αvβ3-specific drugs. In this study I optimized the fermentation culture condition to increase the protein production of Rho mutants expressed in P. pastoris. The protein production of Rho mutants using shake-flask culture is ~100 mg/L. I increased the production of a Rho mutant, HSA-ARLDDL, up to 9-fold with a yield of ~900 mg/L by optimizing pH, temperature, medium composition, and oxygen level of the fermentation process. The final yields of proteins produced from shake-flask and fermentation cultures after purification were 30 mg/L and 170 mg/L, respectively. The recovery of proteins produced from shake-flask and fermentation cultures after purification were 30% and 17%, respectively. It is likely due to higher mis-folding proteins produced from the fermentation process. In our previous study we found that Rho mutants with a 39KKKRTICR47I linker sequence affected their binding affinity and selectivity to integrins α5β1, αvβ3, and αIIbβ3. Therefore, I have successfully expressed three Rho mutants with the 39KKKRT linker sequence (R46A, I47R, and R46A/I47R mutants) and one mutant with the 39SRAGK linker sequence (R46A mutant). The analysis of platelet aggregation and cell adhesion assays showed that the R46A mutant with the 39SRAGK linker sequence exhibited 5.4-, 7.6-, 2.2-, and 5.6-fold decreases in their inhibitory activity to platelet aggregation and integrins αIIbβ3,αvβ3, and α5β1, respectively. In addition, the R46A mutant with the 39KKKRT linker sequence exhibited 4.1-, 24.8-, 1.8-, and 1.8-fold decreases in their inhibitory activity to platelet aggregation and integrins αIIbβ3, αvβ3, and α5β1. In contrast, the I47R mutant with the 39KKKRT linker sequence exhibited 3- and 7-fold increases in their inhibitory activity to integrins αvβ3 and α5β1, respectively. Interestingly, we found that Rho mutant containing the 39KKKRTICARARGDN53P sequence inhibited integrins αIIbβ3, αvβ3, and α5β1 with the IC50 values of 956.0, 5.7, and 6.6 nM, respectively. The finding indicated that this mutant can abolish its activity to integrin αIIbβ3. The mutational study on Rho will provide new insight into design potent integrin-specific disintegrins, and the optimization of fermentation condition of HSA-ARLDDL will serve the basis to produce integrin-specific disintegrins expressed in P. pastoris.
論文目次 目 錄
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
表目錄 IX
圖目錄 X
縮寫檢索表 XII
儀器 XIII

第1章 緒論 1
1-1 背景資料 1
1-1-1 整合蛋白(integrin)之介紹 1
1-1-2 去整合蛋白(disintegrin)之介紹 4
1-1-3 馬來蝮蛇去整合蛋白(Rhodostomin;簡稱Rho)之介紹 6
1-1-4 研究整合蛋白與去整合蛋白結合的重要性與先前研究 7
1-1-5 人血清白蛋白融合馬來亞蝮蛇去整合突變蛋白(Rhodostomin human serum albumin fusion protein;簡稱HSA-Rho)之介紹 8
1-2 酵母菌Pichia pastoris(P. pastoris)表現系統之介紹 9
1-2-1 嗜甲醇酵母菌發酵槽(Pichia pastoris fermentation)條件之介紹 11
1-3 研究動機與目標 12
第2章 材料與方法 14
2-1 發酵槽培養研究 14
2-1-1 實驗菌株、載體、培養基、發酵槽添加物配方 14
2-1-2 發酵槽槽體組裝及系統校正 17
2-1-3 甲醇濃度含量偵測監控 18
2-1-4 以搖瓶培養方式找出最佳pH 值適合發酵槽使用 19
2-1-5 以搖瓶培養方式找出最佳培養配方適合發酵槽使用 19
2-1-6 於甲醇誘導時期加入維他命B群 20
2-1-7 於甲醇誘導時期以1.5 L/min空氣混和1.5 %純氧 21
2-1-8 於甲醇誘導時期以1.5 L/min空氣混和1.5 %純氧並增加甘油磷在培養基為中含量提升為20.4 g/L、36 g/L、48 g/L 22
2-2 利用發酵槽上清液純化HSA-Rho 23
2-3 Rho與突變株重組蛋白之製備 26
2-3-1 實驗菌株、質體與培養基配方 26
2-3-2 重組基因之構築 28
2-3-3 重組蛋白之表現與純化 31
2-3-4 重組蛋白之質譜鑑定 35
2-4 Rho與突變株重組蛋白抑制血小板凝集功能之研究 35
2-4-1 血小板之製備 36
2-4-2 抑制血小板凝集功能之分析 36
2-5 Rho與突變株重組蛋白抑制細胞黏著之研究 37
2-5-1 細胞培養 37
2-5-2 Fibronectin之製備 39
2-5-3 抑制細胞黏著之分析 40

第3章 結果 42
3-1 以搖瓶培養方式找出最佳pH 值 42
3-2 以搖瓶培養方式找出最佳培養配方 42
3-3 於甲醇誘導時期加入維他命B群之影響 42
3-4 甲醇誘導時期以1.5 L/min空氣混和1.5 %純氧培養結果 43
3-5 於甲醇誘導時期混和1.5 L/min空氣及1.5 %純氧延長誘導期並增加磷酸甘油在發酵槽培養基中含量提升為20.4 g/L、36 g/L、48 g/L之結果 43
3-6 發酵槽上清液之純化以製備 HSA-Rho的結果 44
3-7 Rho突變株重組蛋白之製備與鑑定 45
3-8 Rho突變株重組蛋白抑制血小板凝集結果 46
3-9 Rho突變株重組蛋白抑制細胞黏著的結果 46
3-9-1 測試細胞黏著條件的結果 47
3-9-2 Rho突變株重組蛋白對整合蛋白 IIb3之影響 47
3-9-3 Rho突變株重組蛋白對整合蛋白 V3和α5β1之影響 48
3-9-4 對整合蛋白 V3和α5β1兩者皆有特異性之序列整理 48

4章 討論 49
4-1 加入維他命B群、氧氣及提升甘油磷酸的含量使產率上升之原因探討 49
4-2 高於60 g/L 磷酸甘油含量於發酵槽培養基培養可行性探討 49
4-3 發酵槽培養造成錯誤摺疊蛋白可能原因探討 50
4-4 固定發酵槽甲醇濃度同時能將溶氧提升到維持20%可能解決方案 50
4-5 發酵槽溶氧比例與甘油磷酸間的相關性 51
4-6 改善後培養基是否能夠在不同質體構築菌中也能看到效果 51
4-7 提升到克每公升產量之發酵槽層面未來可嘗試方向 51
4-8 Rho與其突變株重組蛋白研究IC46R47I區域對整合蛋白的影響 52
4-9 IC46R47I對整合蛋白辨識所扮演的角色之綜合討論 53
4-10 發酵槽與去整合蛋白研究對未來的貢獻 55
第5章 結論 56
參考文獻 58
表 70
圖 75
附錄 98
自述 110
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