臺灣博碩士論文加值系統

目錄
壹、 前言.......................................................1
一、 研究背景...................................................1
二、 研究目的...................................................2
貳、 文獻回顧...................................................3
一、 核糖核酸酶抑制劑(RNase Inhibitor, RI).......................3
1. RI 簡介......................................................3
2. RI 與 RNase 的結合............................................3
3. RI 在大腸桿菌的表現...........................................4
二、 核糖核酸酶 (Ribonuclease ,RNase)............................4
1. RNase 簡介 ..................................................5
2. 核糖核酸酶 A (RNase A) ......................................5
3. 核糖核酸酶 T1 (RNase T1) ....................................6
4. 核糖核酸酶 H (RNase H).......................................6
三、 大腸桿菌表現系統 ...........................................6
1. 表現載體.....................................................7
2. E. coli BL21(DE3) ..........................................8
四、 分子伴護蛋白 ...............................................8
五、 二硫酥糖醇(Dithiothreitol, DTT) ............................9
六、 發酵技術....................................................9
七、 重組蛋白的誘導方式 .........................................11
1. IPTG 誘導....................................................11
2. 乳糖誘導.....................................................12
參、 實驗架構....................................................13
肆、 實驗方法與材料 .............................................13

一、 實驗材料...................................................14
1. 菌株.........................................................14
2. 載體.......................................................14
3. 培養基.....................................................14
4. 抗生素.....................................................14
5. 標準品.....................................................14
6. 套組........................................................14
7. 化學藥品....................................................15
8. 實驗設備.....................................................16
二、 各式培養基製備 .............................................17
三、 抽取質體 DNA..............................................18
四、 製備瓊脂凝膠 ..............................................19
五、 製備電穿孔之勝任細胞 .......................................19
六、 電穿孔轉形作用 .........................................19
七、 破菌.....................................................20
八、 SDS-PAGE..................................................20
九、 目標 RI 在大腸桿菌中的表現.................................21
1. 最適分子伴護蛋白篩選 .......................................21
2. 誘導溫度....................................................21
3. IPTG 誘導濃度與加入時間點.....................................21
4. 分子伴護蛋白誘導濃度 ....................................22
5. 分子伴護蛋白誘導濃度 ........................................22
6. DTT 最適濃度篩選 .......................................23
7. DTT 最適濃度篩選 ...........................................23
十、 乳糖自誘導 ................................................24

1. 乳糖濃度....................................................24
2. 葡萄糖濃度 ...................................................24
十一、 發酵技術生產 RI...........................................25
1. 批式培養(IPTG 誘導)...........................................25
2. 批式培養(乳糖誘導)............................................25
十二、 目標 RI 純化...........................................26
1. 磁珠純化...................................................26
2. 鎳離子親和性管柱 ............................................27
十三、 重組 RI 的蛋白質定量.....................................28
十四、 重組 RI 濃縮置換保存液....................................28
十五、 活性測定.................................................29
伍、 結果與討論...................................................30
1. 最適分子伴護蛋白篩選 ..........................................30
2. 最適誘導溫度 ..................................................30
3. IPTG 誘導濃度與加入時間點.......................................30
4. 分子伴護蛋白誘導劑濃度 .....................................31
5. DTT 還原劑之最適誘導濃度與添加時間點篩選 .......................32
7. 發酵槽....................................................34
8. RI 之 FPLC 純化與濃縮.....................................35
9. 活性測定...................................................36
陸、 結論.......................................................37
柒、 參考文獻....................................................39
捌、 實驗圖表...................................................46

蘇建豪，2022，由大腸桿菌表現之重組反轉錄酶其生產與保存之最適化條件探討，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
Blackburn, P. (1979). Ribonuclease inhibitor from human placenta: rapid purification and assay. Journal of Biological Chemistry, 254(24), 12484-12487.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), 248-254.
Bruce, D., Cardew, E., Freitag-Pohl, S., & Pohl, E. (2019). How to stabilize protein: stability screens for thermal shift assays and nano differential scanning fluorimetry in the virus-X project. Journal of Visualized Experiments (144), e58666.
Cerritelli, S. M., & Crouch, R. J. (2009). Ribonuclease H: the enzymes in eukaryotes. The FEBS Journal, 276(6), 1494-1505.
Choi, J. H., Keum, K. C., & Lee, S. Y. (2006). Production of recombinant proteins by high cell density culture of Escherichia coli. Chemical Engineering Science, 61(3), 876-885.
Chua, L. H., Tan, S. C., & Liew, M. W. (2018). Process intensification of core streptavidin production through high-cell-density cultivation of recombinant E. coli and a temperature-based refolding method. Journal of Biotechnology, 276, 34-41.
De Vuyst, L., & Vandamme, E. J. (1991). Microbial manipulation of nisin biosynthesis and fermentation. Nisin and Novel Lantibiotics, 397.
Dickson, K. A., Haigis, M. C., & Raines, R. T. (2005). Ribonuclease inhibitor: structure and function. Progress in Nucleic Acid Research and Molecular Biology, 80, 349-374.
Fan, Q., Neubauer, P., & Gimpel, M. (2021). Production of soluble regulatory hydrogenase from Ralstonia eutropha in Escherichia coli using a fed-batch-based autoinduction system. Microbial cell factories, 20(1), 1-12.
Finka, A., Mattoo, R. U., & Goloubinoff, P. (2016). Experimental milestones in the discovery of molecular chaperones as polypeptide unfolding enzymes. Annual Review of Biochemistry, 85, 715-742.
Gay, G., Wagner, D. T., Keatinge-Clay, A. T., & Gay, D. C. (2014). Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae. Plasmid, 76, 66-71.
Glatz, A., Pilbat, A.-M., Németh, G. L., Vince-Kontár, K., Jósvay, K., Hunya, Á., Udvardy, A., Gombos, I., Péter, M., & Balogh, G. (2016). Involvement of small heat shock proteins, trehalose, and lipids in the thermal stress management in Schizosaccharomyces pombe. Cell Stress and Chaperones, 21(2), 327-338.
Gribnau, A. A. M., Schoenmakers, J. G. G., Van Kraaikamp, M., Hilak, M., & Bloemendal, H. (1970). Further studies on the ribonuclease inhibitor from rat liver: stability and other properties. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis, 224(1), 55-62.
Grunberg-Manago, M. (1999). Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annual Review of Genetics, 33(1), 193-227.
Han, X., Ning, W., Ma, X., Wang, X., & Zhou, K. (2020). Improving protein solubility and activity by introducing small peptide tags designed with machine learning models. Metabolic Engineering Communications, 11, 00138.
Johnson, R. J., McCoy, J. G., Bingman, C. A., Phillips Jr, G. N., & Raines, R. T. (2007). Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein. Journal of Molecular Biology, 368(2), 434-449.
Johnson, R. J., Savas, C. J., Kartje, Z., & Hoops, G. C. (2014). Rapid and adaptable measurement of protein thermal stability by differential scanning fluorimetry: updating a common biochemical laboratory experiment. Journal of Chemical Education, 91(7), 1077-1080.
Kaur, J., Kumar, A., & Kaur, J. (2018). Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements. International Journal of Biological Macromolecules, 106, 803-822.
Kim, B. M., Schultz, L. W., & Raines, R. T. (1999). Variants of ribonuclease inhibitor that resist oxidation. Protein Science, 8(2), 430-434.
Kim, H.-K., Rasnik, I., Liu, J., Ha, T., & Lu, Y. (2007). Dissecting metal ion–dependent folding and catalysis of a single DNAzyme. Nature Chemical Biology, 3(12), 763-768.
Kim, Y. E., Hipp, M. S., Bracher, A., Hayer-Hartl, M., & Ulrich Hartl, F. (2013). Molecular chaperone functions in protein folding and proteostasis. Annual Review of Biochemistry, 82, 323-355.
Klink, T. A., Vicentini, A. M., Hofsteenge, J., & Raines, R. T. (2001). High-level soluble production and characterization of porcine ribonuclease inhibitor. Protein Expression and Purification, 22(2), 174-179.
Lopez, P. J., Marchand, I., Joyce, S. A., & Dreyfus, M. (1999). The C‐terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. Molecular Microbiology, 33(1), 188-199.
Malik, K., Matejtschuk, P., Thelwell, C., & Burns, C. J. (2013). Differential scanning fluorimetry: rapid screening of formulations that promote the stability of reference preparations. Journal of Pharmaceutical and Biomedical Analysis, 77, 163-166.
Mamat, U., Wilke, K., Bramhill, D., Schromm, A. B., Lindner, B., Kohl, T. A., Corchero, J. L., Villaverde, A., Schaffer, L., & Head, S. R. (2015). Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microbial Cell Factories, 14(1), 1-15.
Mamat, U., Woodard, R. W., Wilke, K., Souvignier, C., Mead, D., Steinmetz, E., Terry, K., Kovacich, C., Zegers, A., & Knox, C. (2013). Endotoxin-free protein production—ClearColi™ technology. Nature Methods, 10(9), 916-916.
Matthey, B., Engert, A., Klimka, A., Diehl, V., & Barth, S. (1999). A new series of pET- derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins. Gene, 229(1-2), 145-153.
Mitraki, A., & King, J. (1989). Protein folding intermediates and inclusion body formation. Bio/technology, 7(7), 690-697.
Moggridge, J., Biggar, K., Dawson, N., & Storey, K. B. (2017). Sensitive detection of immunoglobulin G stability using in real-time isothermal differential scanning Fluorimetry: Determinants of protein stability for antibody-based therapeutics. Technology In Cancer Research & Treatment, 16(6), 997-1005.
Moreau, M. J., Morin, I., Askin, S. P., Cooper, A., Moreland, N. J., Vasudevan, S. G., & Schaeffer, P. M. (2012). Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. RSC Advances, 2(31), 11892-11900.
Müller-Hill, B. (2011). The lac operon. In The Lac Operon.
Pace, C. N., Heinemann, U., Hahn, U., & Saenger, W. (1991). Ribonuclease T1: structure, function, and stability. Angewandte Chemie International Edition in English, 30(4), 343-360.
Page Sharp, M., Behm, C. A., & Smith, G. D. (1999). Involvement of the compatible solutes trehalose and sucrose in the response to salt stress of a cyanobacterial Scytonema species isolated from desert soils. Biochimica et Biophysica Acta (BBA)-General Subjects, 1472(3), 519-528.
Petsch, D., & Anspach, F. B. (2000). Endotoxin removal from protein solutions. Journal of biotechnology, 76(2-3), 97-119.
Pinsach, J., de Mas, C., & López-Santín, J. (2008). Induction strategies in fed-batch cultures for recombinant protein production in Escherichia coli: application to rhamnulose 1-phosphate aldolase. Biochemical engineering journal, 41(2), 181-187.
Potts, M., Slaughter, S. M., Hunneke, F.-U., Garst, J. F., & Helm, R. F. (2005). Desiccation tolerance of prokaryotes: application of principles to human cells. Integrative and Comparative Biology, 45(5), 800-809.
Raines, R. T. (1998). Ribonuclease a. Chemical reviews, 98(3), 1045-1066.
Restaino, O. F., Cimini, D., De Rosa, M., Catapano, A., & Schiraldi, C. (2011). High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production. Microbial Cell Factories, 10(1), 1-10.
Rodrigues, J. V., Prosinecki, V., Marrucho, I., Rebelo, L. P. N., & Gomes, C. M. (2011). Protein stability in an ionic liquid milieu: on the use of differential scanning fluorimetry. Physical Chemistry Chemical Physics, 13(30), 13614-13616.
Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Communications Biology, 3(1), 1-8.
Šiurkus, J., & Neubauer, P. (2011). Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression. Microbial Cell Factories, 10, 1-11.
Šiurkus, J., & Neubauer, P. (2011). Reducing conditions are the key for efficient production of active ribonuclease inhibitor in Escherichia coli. Microbial Cell Factories, 10(1), 1-15.
Šiurkus, J., Panula-Perälä, J., Horn, U., Kraft, M., Rimšeliene, R., & Neubauer, P. (2010). Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures. Microbial Cell Factories, 9(1), 1-17.
Stathopoulos, C. (1998). Structural features, physiological roles, and biotechnological applications of the membrane proteases of the OmpT bacterial endopeptidase family: a micro-review. Membrane & Cell Biology, 12(1), 1-8.
Suzuki, D. T., & Griffiths, A. J. (1976). An introduction to genetic analysis. WH Freeman and Company.
Taherimehr, Z., Zaboli, M., & Torkzadeh-Mahani, M. (2020). New insight into the molecular mechanism of the trehalose effect on urate oxidase stability. Journal of Biomolecular Structure and Dynamics, 1-11.
Tereshina, V. (2005). Thermotolerance in fungi: the role of heat shock proteins and trehalose. Microbiology, 74(3), 247-257.
Thiry, M.,Cingolani, D. (2002). Optimizing scale-up fermentation processes.TRENDS in Biotechnology, 20(3), 103-105.
Thomas, J. G., Ayling, A., & Baneyx, F. (1997). Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. Applied Biochemistry and Biotechnology, 66(3), 197-238.
Walter, S., & Buchner, J. (2002). Molecular chaperones—cellular machines for protein folding. Angewandte Chemie International Edition, 41(7), 1098-1113.
Waterman, K. C. (2009). Understanding and predicting pharmaceutical product shelf-life. Handbook of Stability Testing in Pharmaceutical Development: Regulations, Methodologies, and Best Practices, 115-135.
Xin, F., Dong, W., Dai, Z., Jiang, Y., Yan, W., Lv, Z., ... & Jiang, M. (2019). Biosynthetic technology and bioprocess engineering. In Current Developments in Biotechnology and Bioengineering (pp. 207-232). Elsevier.
Zhang, J., Suflita, M., Fiaschetti, C., Li, G., Li, L., Zhang, F., Dordick, J., & Linhardt, R. (2015). High cell density cultivation of a recombinant Escherichia coli strain expressing a 6‐O‐sulfotransferase for the production of bioengineered heparin. Journal of Applied Microbiology, 118(1), 92-98.
Zhou, Y., Ma, X., Hou, Z., Xue, X., Meng, J., Li, M., Jia, M., & Luo, X. (2012). High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production. Protein Expression and Purification, 85(1), 38-43.