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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃慶璨 | |
dc.contributor.author | Yu-Jiun Lin | en |
dc.contributor.author | 林俞均 | zh_TW |
dc.date.accessioned | 2021-05-13T08:35:52Z | - |
dc.date.available | 2018-08-25 | |
dc.date.available | 2021-05-13T08:35:52Z | - |
dc.date.copyright | 2016-08-25 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-18 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/3673 | - |
dc.description.abstract | T4 endonuclease V (T4N5)是T4 bacteriophage中的DNA修復酵素,為大小16 kDa的蛋白質,可專一性辨認因紫外線造成DNA損傷而形成的環丁烷嘧啶二聚體(cyclobutane pyrimidine dimer, CPD)並切除,再由後續DNA修復機制進行修補。過去研究利用大腸桿菌生產重組T4N5,結合微脂體(Liposome)技術製成乳劑,針對缺乏核苷酸切除修復(nucleotide excision repair)能力的患者進行臨床實驗,發現可減少病灶生成,顯示T4N5移除紫外線造成的DNA損傷功能對人體亦有所幫助。為使此技術能廣泛應用於防曬乳及保養品中,提升產品附加價值,須尋求低成本大量生產重組T4N5的方法。現行的重組T4N5多以大腸桿菌進行生產,然而欲取得目標蛋白質須經破菌及多道純化手續,且有內毒素的疑慮。嗜甲醇酵母菌Pichia pastoris為單細胞真菌表達系統,可進行高密度培養,生產基因重組蛋白質成本相較低廉,同時沒有內毒素疑慮並可利用外泌訊息胜肽將目標蛋白質分泌至培養基中以方便純化。本研究利用嗜甲醇酵母菌P. pastoris KM71H作為重組T4N5表達系統,以搖瓶和醱酵槽進行培養,添加甲醇作為碳源並誘導異源蛋白質表現。搖瓶培養可成功外泌表現出序列正確且具活性的T4N5,活性可達0.47±0.05 U/µL。但以醱酵槽進行高細胞密度培養時,發現以2.6 mL/L/h的速率添加甲醇可能會對菌體造成壓力使部分菌體死亡並裂解釋放胞內蛋白酶,但減低甲醇添加量又不利於菌體生產異源蛋白質,產量皆不理想,須以其他方式減少菌體死亡裂解及抑制蛋白酶作用。本研究發現,同樣以約2.6 mL/L/h的速率添加甲醇,將誘導溫度由30oC降低至 23oC,可減低培養基中的蛋白酶含量,並增進甲醇異化代謝路徑,大幅提升目標蛋白質表現量。 | zh_TW |
dc.description.abstract | T4 endonuclease V (T4N5) is a DNA repair enzyme isolated from T4 bacteriophage. It can specifically recognize and remove cyclobutane pyrimidine dimer which is resulted from the UV-induced DNA damage and might cause skin cancer. In previous reports, in conjunction of recombinant T4N5 expressed by Escherichia coli and liposome, T4N5 liposome lotion effectively penetrated into skin in vivo and clinical trials also indicated its potential for UV-induced DNA damage treatment. In order to extensive application of T4N5 in skin care products such as sunscreen, it’s important to develop a method for large-scale production recombinant T4N5 with low expenses. The major recombinant T4N5 are currently produced by E. coli. However, the procedures include cell lysis and protein purification, which may increase the production cost. Moreover, it is at risk due to endotoxins. Pichia pastoris is a well-defined expression system which can be cultivated at high cell density at low cost. In this study, P. pastoris KM71H was used as a heterologous expression system, and methanol was added to induce recombinant T4N5 expression. In flask, we have successfully expressed extracellular T4N5 with correct conformation and function. The T4N5 activity in supernatant of flask culture reached up to 0.47±0.05 U/µL. When using 5 L fermenter, methanol fed at 2.6 mL//L/h induced cell lysis and release of cellular proteases, while reducing methanol feeding rate led to low yield of target protein yield. Therefore, it’s necessary to modify other induction conditions. In this study, we found that reduction of the induction temperature from 30oC to 23oC improved recombinant protein yield remarkably at the same methanol feeding rate. The amount of proteases in the culture decreased, and methanol dissimilation is improved. | en |
dc.description.provenance | Made available in DSpace on 2021-05-13T08:35:52Z (GMT). No. of bitstreams: 1 ntu-105-R03b22022-1.pdf: 2324613 bytes, checksum: 3f207acc45b3e9d00f17c1652de20065 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 目錄
摘要 I Abstract II 圖目錄 V 表目錄 VI 第一章、 前言 1 一、 T4 endonuclease V 1 1. 結構及作用機制 1 2. 應用價值 2 3. 生產現況 3 二、微生物異源表達系統 4 1. 原核生物表達系統 4 2. 真核生物表達系統 4 三、Pichia pastoris表達系統 7 1. 酒精氧化酶及其啟動子 7 2. 菌株與甲醇表現型 8 3. 訊息胜肽 8 4. 大規模表現 9 四、研究動機與目的 14 第二章、 材料與方法 17 一、 實驗菌株與培養條件 17 1. Escherichia coli 17 2. Pichia pastoris 17 二、培養基與藥品 17 三、Pichia pastoris表現載體建構 20 四、Pichia pastoris轉形與篩選 23 1. 轉形DNA製備 23 2. 勝任細胞製備 23 3. 電穿孔轉形[58] 23 4. Pichia pastoris轉形株篩選 24 五、Pichia pastoris誘導表現T4 endonuclease V 25 1. 搖瓶培養 25 2. 醱酵槽培養 25 六、蛋白質產物分析 27 1. 西方墨點法 27 2. LC-MS/MS 27 七、T4 endonuclease V活性測定 30 八、培養基胞外上清液蛋白酶活性分析 31 第三章、 結果 32 一、 轉形株篩選與確認 32 1. 抗性濃度梯度培養基篩選轉形株 32 2. 染色體PCR確認目標基因插入 32 二、 搖瓶培養結果 35 1. 以SDS-PAGE篩選表現量高轉形株 35 2. 以西方墨點法確認目標蛋白質順利表現 35 3. LC-MS/MS確認目標蛋白質 35 4. 搖瓶上清液T4N5活性測定 35 三、 醱酵槽培養結果 40 1. 以BSM培養 40 2. 以BMGY置換到BMMY培養 40 3. 蛋白酶活性分析 41 第四章、 討論 51 一、 培養基成分之探討 51 1. BSM (Fermentation Basal Salts medium) 51 2. BMGY培養,誘導前置換為BMMY 52 二、 甲醇添加速率之探討 52 1. 鹼添加量 52 2. OD600及生菌數 53 3. 胞外上清液蛋白酶活性 53 4. SDS-PAGE分析及胞外上清液總可溶蛋白質 53 三、 其他培養條件之探討 54 第五章、 結論 56 第六章、 未來展望 57 第七章、 參考文獻 58 | |
dc.language.iso | zh-TW | |
dc.title | 嗜甲醇酵母菌Pichia pastoris醱酵生產重組T4 endonuclease V之研究 | zh_TW |
dc.title | The production of recombinant T4 endonuclease V in Pichia pastoris fermentation | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李昆達,楊啟伸,吳?承 | |
dc.subject.keyword | 重組T4 endonuclease V,Pichia pastoris,醱酵培養,甲醇誘導,降溫培養, | zh_TW |
dc.subject.keyword | Recombinant T4 endonuclease V,Pichia pastoris,Fermentation,Methanol induction,Low-temperature cultivation, | en |
dc.relation.page | 65 | |
dc.identifier.doi | 10.6342/NTU201603209 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2016-08-20 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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