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研究生:黃啟田
研究生(外文):Chi-tien Huang
論文名稱:基因重組his-tagstreptavidin生產製程之研究
論文名稱(外文):Study on the production process of the recombinant his-tag streptavidin
指導教授:劉仲康劉仲康引用關係
指導教授(外文):Jong-Kang Liu
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:82
中文關鍵詞:卵白素his-tag鏈黴卵白素純化最適條件
外文關鍵詞:purificationhis-tag streptavidinAvidinoptimal conditions
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本研究中以E. coli BL21(DE3) 作為表達重組蛋白his-tag streptavidin的菌株,從培養條件、培養基組成、誘導條件及誘導時機等研究中,尋找出表現重組蛋白的最佳製程,另外在純化製程上探討疏水性管柱及親和性管柱在純化上的差異,以及熱處理粗萃液的效果。結果發現以LB培養基培養時,最佳表現重組蛋白的條件為培養溫度37℃,培養pH 6.0 - 7.0,誘導溫度為37℃,誘導時期在對數期的晚期或靜止期開始時 (OD600值介於1.1 - 1.8之間),誘導之IPTG濃度為0.1 mM,以2 mM乳糖取代也可得到相同結果,另外最佳的培養基配方為TB,以5公升醱酵在較佳培養及誘導絛件下,所得到重組蛋白之最高產量為81.1 mg/L。在純化製程上,以親和性管柱來純化,可得到高純度的重組蛋白,活性可達14 U/mg以上,回收率高達97%,而比較疏水性管柱純化回收率則只有51%。另外若以75℃加熱10分鐘來處理粗萃液,比活性也可達到14.1 U/mg,不過回收率則降為64%。
In this study, we used E. coli strain BL21 (DE3) to express the recombinant protein his-tag streptavidin. To find out the optimal production conditions, we studied on the culture conditions, medium composition, induction conditions and the timing of induction. In the purification processes we tried to find out the difference between hydrophobic column and affinity column. We also tested the effect of heat treatment on the crude extract to increase the recombinant protein yield. The results showed that when cultured in LB medium, the optimal culture conditions of recombinant protein expression are 37°C, pH 6.0 to 7.0, and the induction temperature is 37°C. The best induction time is at late log phase or the early stationary phase when OD600 values reached to the ranged of 1.1 to 1.8. The inducer, IPTG concentration is 0.1 mM, which can also replaced with 2 mM lactose. The best production medium is TB medium. When cultured in 5 liters fermentor with optimal culture and induction condition, the highest recombinant protein yield could be 81.1 mg /L. To improve the purification process, we used a affinity chromatography. The purified high homogeneous recombinant protein had a high biotin binding activity up to 14 U / mg, and the recovery yield could be as high as 97% in comparing with the hydrophobic column was only 51%. When we treated the crude extract with 75 ℃ for 10 min, the biotin binding activity was 14.1 U / mg, but the recovery rate decreased to 64 %.
中文摘要 ………………………………………………………………………….. Ⅰ
英文摘要 ………………………………………………………………………….. Ⅱ
致謝 ……………………………………………………………………………….. Ⅲ
目錄 …………………………………………………………………………………Ⅳ
圖、表索引…………………………………………………………………………. Ⅵ
第一章 前言
1.1 Streptavidin ……………………………………………….…………………. 1
1.2 Streptavidin的應用…………………………………….…………………..... 3
1.3 Streptavidin 的生產製程……………………………………..……………... 4
1.4以lac promoter表現外來蛋白之影響因子………………….……………... 8
1.5 研究目的 ..………………………………………………………………….. 11
1.6 論文架構 .…………………………………………………………….…….. 12
第二章 材料與方法
2.1 實驗材料與培養基………………………………………………………….. 13
2.2 實驗方法
2.2.1. 以S. avidinii生產SA ………………………….……………………. 14
2.2.2 確認E. coli (FE66) 是否表現his-tag SA……………………………. 14
2.2.3 E. coli (FE66)最佳生長條件之研究……………….…………………. .14
2.2.4 細胞破碎方法………………………………………………………… .15
2.2.5 菌體乾重之估算方法 ……………………………………..………… .15
2.2.6 蛋白質快速檢測 (Bio-Rad Protein Assay - Standard Procedure)……..15
2.2.7 SA活性 (binding biotin activity)之分析方法………………………….16
2.2.8 粗萃液中SA活性之估算方式………………………………………...18
2.2.9 建立SA活性分析方法範圍…………………………………………...18
2.2.10 SA含量之計算方式………………………………………………......18
2.2.11 最佳his-tag SA表現條件之研究………………………………....….18
2.2.12 不同培養基對E. coli (FE66)生長及重組蛋白表現量的差異……....19
2.2.13 5公升醱酵槽培養測試……………………………………………...20
2.2.14 SDS-PAGE電泳分析………………………………………………….20
2.2.15 His-tag SA純化試驗…………………………………………………..22
2.2.16 比較不同溫度加熱處理SA粗萃液的影響………………………….24
第三章 結果
3.1 以S. avidinii生產SA……………………………………….….….…....…. 25
3.2 確認E. coli (FE66) 是否表現his-tag SA……………………………….…25
3.3 宿主E. coli (FE66)最佳生長條件之研究……………………………….….25
3.4 以IPTG誘導his-tag SA之最佳表現條件研究……………………...……25
3.5 不同培養基對E. coli (FE66) 生長及SA表現量的差異………………….26
3.6 E. coli (FE66) 5公升醱酵槽培養測試………………………………………27
3.7 SA活性分析方法範圍……………………………………………………….28
3.8 純化試驗……………………………………………………………………..28
3.9 不同溫度加熱處理粗萃液…………………………………………………..29
第四章 討論 ………………………………………………………………..…… 30
參考資料 ………………………………………………………………………….. 34
圖表 ……………………………………………………………………………….. 39
附錄 ……………………………………………………………………………….. 65

圖、表索引

表1. 不同生長期加入IPTG對E. coli (FE66)表達SA之影響…………………..39
表2. 不同溫度環境下誘導對E. coli (FE66)表達SA之影響……………………..39
表3. 不同IPTG濃度誘導SA表現…………………………………………………40
表4. 誘導時以IPTG或乳糖作為誘導物對SA產率之差異………………………40
表5. 不同培養基對SA表現量的差異……………………………….…..………..41
表6. 以5公升醱酵槽培養,不同的培養基培養下所得到SA產率……………..41
表7. 測定SA活性分析方法之可信範圍…………………………………………..42
表8. 硫酸銨沉澱處理SA粗萃液…………………………………………………..42
表9. 疏水性管柱(HiTrap Phenyl HP)純化………………………………………....43
表10. 親和性管柱(Histrap FF)純化………………………………………………..43
表11. 親和性管柱(His Trap FF, 1 mL)的capacity…………………………………44
表12. 不同溫度加熱處理SA粗萃液……………………………………………...44
表13. 本研究之his-tag SA與市面上產品之比較…………………………………45
圖1. 以搖瓶培養S. avidinii,在不同培養時間所測得SA分泌量的差異……….46
圖2. S. avidinii之培養液經硫酸銨沉澱回溶之SDS-PAGE…………………….47
圖3. 比較E. coli (BL21)與E. coli (FE66)粗萃液之SDS-PAGE結果…………….48
圖4. 不同溫度下培養之E. coli (FE66)生長曲線………………………………….49
圖5. 不同pH培養之E. coli (FE66)生長曲線…………………………………….50
圖6. 不同生長期誘導E.coli (FE66)表現his-tag SA之差異……………………...51
圖7. 不同培養基培養E. coli (FE66)之生長曲線.... ... ... ... ... ... ... ... ... .. ... ... ... .52
圖8. 不同培養基培養E.coli (FE66)並表現SA之差異(1)………………………..53
圖9. 不同培養基培養E.coli (FE66)並表現SA之差異(2)………………………..54
圖10. 以5公升醱酵槽測試LB培養之E. coli (FE66)生長曲線…………………55
圖11. 以5公升醱酵槽測試TB培養之E. coli (FE66)生長曲線…………..…….56
圖12. 以5公升醱酵槽測試無機氮源配方培養之E. coli (FE66)生長曲線.…….57
圖13. 以5公升醱酵槽饋料培養之E. coli (FE66)生長曲線……………….…….58
圖14. 菌液OD600值與菌體乾重 (DCW)之迴歸曲線……………………………59
圖15. SA活性分析方法之可信範圍……………………………………….…….60
圖16. 以疏水性管柱Hitrap Phenyl HP純化之層析圖譜…………………………61
圖17. 以親和性管柱Histrap FF純化之層析圖譜…………………………………62
圖18. 親和性管柱Histrap FF純化後之SDS-PAGE結果…………………………63
圖19. 不同的處理溫度下的SDS-PAGE結果……………………………………..64
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