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博碩士論文 etd-0214108-144012 詳細資訊
Title page for etd-0214108-144012
論文名稱
Title
基因重組his-tag streptavidin生產製程之研究
Study on the production process of the recombinant his-tag streptavidin
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
82
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2007-12-26
繳交日期
Date of Submission
2008-02-14
關鍵字
Keywords
卵白素、his-tag鏈黴卵白素、純化、最適條件
purification, his-tag streptavidin, Avidin, optimal conditions
統計
Statistics
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The thesis/dissertation has been browsed 5661 times, has been downloaded 6362 times.
中文摘要
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%。
Abstract
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 %.
目次 Table of Contents
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
參考文獻 References
Aldwin L, R Toso, R Goodson, and J Hunter. 1990 Improvement of production, assay and purification of streptavidin. J Ind Microbiol 5: 239-246.
Argarana CE., ID Kuntz, S Birken, R Axel, and CR Cantorl. 1986 Molecular cloning and nucleotide sequence of the streptavidin gene. Nucleic Acids Res 14: 1871-1882.
Auger EA, and GN Bennett. 1987. Temperature optimization of in vivo expression from the E. coli trp and trp: lac promoters. Biotechnol Lett 8: 157-162.
Bayer EA, H Ben-Hur, and M Wilchek. 1990 Isolation and properties of streptavidin. Methods Enzymol 184: 80-89.
Braun P, G Gerritse, JM van Dijl, and WJ Quax. 1999 Improving protein secretion by engineering components of the bacterial translocation machinery. Curr Opin Biotechnol. 10:376-381
Cabilly S. 1989 Growth at sub-optimal temperature allows the production of functional, antigen-binding Fab fragments in Escherichia coli. Gene 85: 553-557.
Cazin J, M Suter, and JE Butler. 1988 Production of streptavidin in a synthetic medium. J Immunol Methods 113: 75-81.
Clare DA, VW Valentine, GL Catignani, and HE Swaisgood. 2001 Molecular design, expression, and affinity immobilization of a trypsin-streptavidin fusion protein. Enzyme Microb Technol 28: 483-491.
De Bellis D, and I Schwartz. 1990 Regulated expression of foreign genes fused to lac: control by glucose levels in growth medium. Nucleic Acids Res 18: 1311.
Diamandis EP, and TK Christopoulos. 1991 The biotin – (strept)avidin system : principles and applications in biotechnology. Clin Chem 37: 625-638.
Donovan RS, CW Robinson, and BR Glick. 1996 Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J Ind Microbiol 16: 145-154.
Gallizia A, C Lalla, E Nardone, P Santambrogio, A Brandazza, A Sidoli, and P Arosio . 1998 Production of a soluble and functional recombinant streptavidin in Escherichia coli. Protein Expr Purif 14: 192-196.
Glick BR. 1995 Metabolic load and heterologous gene expression. Biotechnol Adv 13: 247-261.
Green NM. 1964 The molecular weight of avidin. Biochem J 92: 16-17.
Green NM. 1965 A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem J 94: 23-24.
Green NM. 1970 Spectrophotometric determination of avidin and biotin. Meth Enzymol 18: 418.
Green NM, and EJ Toms. 1972 The dissociation of avidin-biotin complexes by guanidinium chloride . Biochem J 130: 707-711.
Green NM. 1990 Avidin and streptavidin. Methods Enzymol. 184: 51-67.
Halling SM, FJ Sanchez-Anzal, R Fukuda, RH Doi, and CF Meares. 1977 Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis. Biochemistry. 16: 2880-2884.
Hiller Y, EA Bayer, and M Wilchek. 1990 Nonglycosylated avidin. Methods Enzymol 184: 68-70.
Horn U, W Strittmatter, A Krebber, U Knupfer, M Kujau, R Wenderoth, K Muller, S Matzku, A Pluckthun, and D Riesenberg. 1996 High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions. Appl Microbiol Biotechnol. 46: 524-532
Huang TS, and RJ DeLange. 1971 Egg white avidin. II. Isolation, composition, and amino acid sequences of the tryptic peptides. J Biol Chem 246: 686-697.
Karp M, and CO Blom. 1999 A streptavidin–luciferase fusion protein: comparisons and applications. Biomol Eng 16: 101-104.
Korz DJ, U Rinas, K Hellmuth, EA Sanders, and WD Deckwer. 1995 Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol. 39:59-65
Melamed MD, and NM Green. 1963 Avidin. 2. Purification and composition. Biochem J 89: 591-599.
Nagarajan V, R Ramaley, H Albertson, and M Chen. 1993 Secretion of streptavidin from Bacillus subtilis. Appl Environ Microbiol 59: 3894-3898.
Nakano K, M Rischke, S Sato, and H Markl. 1997 Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor. Appl Microbiol Biotechnol. 48:597-601
Neubauer P, K Hofmann, O Holst, B Mattiasson, and P Kruschke. 1992 Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer. Appl Microbiol Biotechnol 36: 739-744.
Persson R, J Nord, R Roth, and PO Nyman. 2002 dUTPase from Escherichia coli; high-level expression and one-step purification. Prep Biochem Biotechnol. 32: 157-172
Sambrook, and Russell. 2001 Molecular cloning (a laboratory manual). 3rd edition, Volume 3: A2.2~2.4.
Sano T, and CR Cantor. 1990 Expression of a cloned streptavidin gene in Escherichia coli. Proc Natl Acad Sci USA 87: 142-146.
Sharma SS, S Chong, and SW Harcum. 2006 Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli. J Biotechnol 125: 48-56.
Shiloach J, and R Fass. 2005 Growing E. coli to high cell density – a historical perspective on method development. Biotechnol Adv 23: 345-357.
Sorensen HP, HU Sperling-Petersen, and KK Mortensen. 2003 A favorable solubility partner for the recombinant expression of streptavidin. Protein Expr Purif 32: 252-259.
Sorensen HP, HU Sperling-Petersen, ang KK Mortensen. 2003 Dialysis strategies for protein refolding: preparative streptavidin production. Protein Expr Purif. 31: 149-154.
Stapley EO, JM Mata, IM Miller, TC Demny, and HB Woodruff. 1963 Antibiotic MSD-235. I. Prodduction by Streptomyces avidinii and Streptomyces lavendulae. antimicrobial agents chemother (bethesda). 161: 20-27
Suter M, J Cazin, JE Butler, and DM Mock. 1988 Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium. J Immunol Methods 113: 83-91.
Tausig F, and FJ Wolf. 1964 Streptavidin – a substance with avidin-like properties produce by microorganisms. Biochem Biophys Res Commun 14: 205-209.
Voss S, and A Skerra.1997 Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the strep-tag II peptide and improved performance in recombinant protein purification. Protein Eng. 10:975-982
Wei RD, and LD Wright. 1964 Heat stability of avidin and avidin-biotin complex and influence of ionic strength on affinity of avidin for biotin. Proc Soc Exp Biol Med 117: 341.
Wilchek M., and EA Bayer. 1990 Introduction to avidin-biotin technology. Methods Enzymol 184: 5-13.
Wong SL.1995 Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr Opin Biotechnol. 6: 517-522
Wu SC, MH Qureshi, and SL Wong. 2002 Secretory production and purification of functional full-length streptavidin from Bacillus subtillis. Protein Expr Purif 24: 384-356.
Wu SC, and SL Wong. 2005 Engineering soluble monomeric streptavidin with reversible biotin binding capability. J Biol Chem. 280: 23225-23231.
Wu SC, and SL Wong. 2006 Intracellular production of a soluble and functional monomeric streptavidin in Escherichia coli and its application for affinity purification of biotinylated proteins. Protein Expr Purif. 46: 268-273.
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