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研究生:許紹彥
研究生(外文):Hsu, Shaoyen
論文名稱:於大腸桿菌中利用σ32因子與目標蛋白質共表達來改善重組酵素蛋白質的可溶性及活性
論文名稱(外文):Improve solubility and activity of a recombinant enzyme protein by co-expressing sigma 32 factor in Escherichia coli
指導教授:李文乾
指導教授(外文):Lee, Wenchien
口試委員:黃光策劉懷勝
口試委員(外文):Huang, kuangtseLiu, Hwaishen
口試日期:2012-07-02
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:164
中文關鍵詞:sigma 32因子熱休克轉錄因子N-乙醯-D-神經胺糖酸醛縮酶共表達包涵體構形品質
外文關鍵詞:sigma 32heat shock transcription factorN-acetyl-D-neuraminic acid aldolaseco-expressioninclusion bodyconformational quality
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  本研究使用大腸桿菌BL21過量表現融合酵素蛋白質N-乙醯-D-神經胺糖酸醛縮酶(Neu5Ac aldolase),其中,酵素之N端連接麩胱苷肽-S轉移酶蛋白(GST, Glutathione S-transferase),C端融合五個精胺酸(5R, 5 Arginine)形成雙標誌酵素蛋白質。實驗上利用基因重組方法將實驗室現有的菌種E. coli BL21 pGEX-2TK-nana-5R加入新切位成為E. coli BL21 pGEX-2TK-nana-5R-XhoI(簡稱XhoI,控制組),再將表現σ32因子的基因(rpoH)嵌入pGEX-2TK質體中,接於Neu5Ac aldolase序列後面以shine-dalgrno ribosome binding site連結成為E. coli BL21 pGEX-2TK-nana-5R-rpoH(簡稱rpoH,實驗組),額外比較一株實驗組的雙質體菌種E. coli BL21 pGEX-2TK-nana-5R & pBAD33-rpoH(簡稱dual,實驗組),分別加入異丙基-D-硫代半乳糖(IPTG)或阿拉伯糖(arabinose)誘導後共表達,進一步去探討蛋白質體變化以及目標酵素活性與溶解度。嵌入rpoH基因主要是希望σ32因子以過量表達的方式代替熱休克這類的應力條件來間接促進下游的熱休克蛋白質(輔酶)表現,藉此方法以補強大腸桿菌表現外源蛋白時,會產生包涵體缺點的不足進而使外源蛋白的溶解度及活性能有所提升,使得工業化量產酵素時,能夠有效地降低製程過程中的成本。
本實驗研究以二維膠體電泳探討上述三株菌種誘導前後的熱休克蛋白與σ32因子之間的蛋白質體變化,發現XhoI、rpoH以及dual之菌種於誘導三小時後,小熱休克蛋白ibpA/B分別有顯著上升(p-value < 0.05); rpoH比起XhoI於誘導三小時後,小熱休克蛋白ibpA/B分別有顯著下降(p-value < 0.05)。
  在西方點墨法實驗中顯示XhoI在誘導後1、2及3小時比起未誘導時,σ32因子的表現量提升了3.5、2.1以及2.1倍,dual則提升了12.6、11以及12.8倍,rpoH提升了15.3、16.4以及19.1倍;在溶解度方面,rpoH以及dual比起XhoI於誘導1小時的溶解度有明顯上升至27%以及10% ( p-value < 0.05),但隨著誘導時間增長而下降。
  在活性實驗方面,顯示rpoH比起XhoI在誘導後1、2及 3小時的全細胞比活性分別提升了1.6、1.42、以及1.41倍,dual比起XhoI提升了3.23、2.63以及2.29倍;菌體破萃後上清液比活性rpoH比起XhoI提升了1.6、3以及2.5倍,dual比起XhoI則是提升了2、1.5以及1.7倍;而剩下不溶體回溶液的比活性rpoH比起XhoI則提升了1.4、1.8、2.4倍,dual則比XhoI提升了5、4以及2.7倍。
  因此本實驗研究證實含有rpoH基因共表達之菌株,所生產出來的目標酵素蛋白質有較好的溶解度以及活性,與一開始的假設一致。
  We used Escherichia coli BL21 overexpressing recombinant enzyme protein, N-acetyl-D-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R or Neu5Ac aldolase), this enzyme protein overexpressed with glutathione S-transferase (GST, Glutathione S-transferase) and polyionic peptide (5R, 5 Arginine) tags became two tags fusion enzyme protein. By genetic engineering technique, we add a new cutting site into the existed E. coli strain (E. coli BL21 pGEX-2TK nana5R), to become E. coli BL21 pGEX-2TK nana5R-XhoI (abbr. XhoI). At the same time we add σ32 factor gene (rpoH) into pGEX-2TK with shine-dalgrno ribosome binding site, right after Neu5Ac aldolase sequence, to become E. coli BL21 pGEX-2TK nana5R-rpoH (abbr. rpoH). During E. coli culture, we add Isopropyl β-D-1-thiogalactopyranoside (IPTG) or arabinose to induce these engineered enzymatic protein expression. Compare with E. coli BL21 pGEX-2TK nana5R and pBAD33 rpoH (abbr. dual, control group), we studied for the changes of proteomics and the activity or solubility of target enzymatic protein. We add rpoH gene to overexpressing σ32 factor, that offsets the conditions of heat-shock. In this condition, E. coli will express heat-shock proteins to improve the defects of low solubility and activity of overexpressed proteins while inclusion body forming. This way will reduce the cost of artificial protein production.
  Then we extract the protein of E. coli of each group. Through 2-dimensional SDS-PAGE to study the proteomics changes of heat-shock proteins and σ32 factor. The results indicated that XhoI, rpoH and dual groups showed a significant increase of ibpA/B after 3 hours of IPTG or arabinose induction compare with control group (p < 0.05). And a significant decrease of ibpA/B protein in rpoH group compare with XhoI group after 3 hours of IPTG induction (p < 0.05).
  In western blot, that also indicated σ32 protein increased 3.5 times, 2.1 times and 2.1times in XhoI group after 1, 2, 3 hours of IPTG induction compare with 0 hour. And σ32 protein increased 12.6 times, 11 times and 12.8times in dual group after 1, 2, 3 hours of IPTG and arabinose induction compare with 0 hour. In rpoH group, σ32 protein increased 15.3 times, 16.4 times and 19.1 times after 1, 2, 3 hours of IPTG or arabinose induction compare with 0 hour. The solubility of Neu5AC aldolase had a significant 27% increase in rpoH group compare to XhoI group after 1 hour of IPTG induction, but decrease with induction time. The solubility of Neu5AC aldolase had a significant 10% increase in dual group compare to XhoI group after 1 hour of IPTG and arabinose induction, but decrease with induction time.
  Another results showed that whole cell activity in rpoH group increased 1.6 times, 1.42 times and 1.41 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And the whole cell activity in dual group also increased 3.23 times, 2.63 times and 2.29 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group. The specific activity of supernatant after lysis in rpoH group increased 1.6 times, 3 times and 2.5 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And the specific activity of dual group also increased 2 times, 1.5 times and 1.7 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group. The specific activity in the rest insoluble in rpoH group increased 1.4 times 1.8 times and 2.4 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And in dual group also increased 5 times, 4 times and 2.7 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group.

目錄
中文摘要
Abstract
目錄
圖目錄
表目錄
縮寫對照表
第一章 緒論
1.1 前言
1.2 Escherichia coli
1.3 包涵體
1.4 轉錄因子σ32
1.5 N-acetyl-D-neuraminic acid aldolase
1.6 蛋白質體學
1.6.1 二維膠體電泳(2-DE)
1.6.2 西方點墨法(Western Blot)
1.7 基因重組大腸桿菌的醱酵
1.7.1 甘油為培養基高細胞密度培養大腸桿菌
1.8 研究目標
第二章 實驗藥品
2.1 基因重組
2.2 重組菌培養、誘導及破萃
2.3 蛋白質定量
2.4 SDS-PAGE
2.5 二維膠體電泳(2-DE)
2.6 西方點墨法(Western Blot)
2.7 活性測定
2.8 scanning electron microscope (SEM)
2.9 甘油高密度醱酵
第三章 實驗儀器
3.1 基因重組
3.2 重組菌培養相關儀器
3.3 蛋白質定量
3.4 SDS-PAGE
3.4 二維膠體電泳(2-DE)
3.5 西方點墨法(western blot)
3.6 活性測定
3.7高細胞密度醱酵
3.8 scanning electron microscope (SEM)
3.9 Acrylamide Quenching量測
第四章 實驗步驟與分析
4.1 實驗流程
4.2 基因重組
4.2.1 菌種來源與質體建構
4.2.2 質體抽取與寡核酸引子(primer)設計
4.2.3 聚合酶連鎖反應(Polymerase Chain Reaction, PCR)
4.2.4 DNA瓊脂凝膠電泳分析
4.2.5 PCR產物純化
4.2.6 DNA片段純化
4.2.7 限制酶切反應(digestion)
4.2.8 黏合反應(ligation)
4.2.9 勝任細胞(competent cell)的製備
4.2.10 熱休克轉型法
4.2.11 重組菌的培養與凍存
4.3 蛋白質定量
4.4 SDS-PAGE
4.5 二維膠體電泳(2-DE)
4.5.1 胞內蛋白質前處理
4.5.2回溶蛋白質等電點分離(IEF)
4.5.3 SDS-PAGE
4.5.4 銀染法
4.6 西方點墨法(Western Blot)
4.7 全細胞比活性測定
4.8 supernatant及pellet比活性測定
4.9 溶解度分析
4.10 以SEM (Scanning Electron Microscope)觀察不溶體外觀
4.11 Acrylamide Quenching量測
4.12 高細胞密度甘油醱酵
第五章 實驗結果與討論
5.1 基因重組
5.2 LB搖瓶培養
5.2.1 生長曲線
5.2.2 目標蛋白質表現
5.2.3 WB驗證轉錄因子σ32的身分及表達
5.2.4 目標蛋白質溶解度分析
5.2.5 以2-DE分析誘導前後HSPs之表現
5.2.6 全細胞比活性
5.2.7 可溶性蛋白質與不溶體比活性
5.2.8 誘導前後不溶體外觀
5.3 Acrlamide Quenching量測
5.4 以甘油為碳源高密度醱酵
5.4.1 生長曲線
5.4.2 高細胞密度培養下每升凍乾菌粉重估計
5.4.3 目標蛋白質表現
5.4.4 目標蛋白質誘導破萃後的表現
5.4. 以甘油為碳源做高細胞密度醱酵之全細胞比活性比較
5.4.4以2-DE分析HSPs於誘導前後之關聯
第六章 結論與建議
6.1 結論
6.2 建議
參考文獻
附錄A 重組菌定序結果
附錄B 西方點墨法(σ32)重複實驗結果
附錄C 二維膠體電泳重複實驗
附錄D 全細胞活性p-value
附錄E 溶解度表現
附錄F 以LB培養基培養重組菌株之菌粉乾重與O.D值做圖

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