Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3154
標題: 不同宿主細胞對INP-INT膜上表現系統生產EGFP之影響
Comparison of INP-INT surface display system in various E. coli host cells for EGFP production
作者: 陳啟銘
Chen, Chi-Ming
關鍵字: 重組蛋白;Recombinant protein;冰核蛋白;內含子;綠螢光蛋白;細胞表面表現;Surface display;Ice nucleation protein;Intein;EGFP
出版社: 化學工程學系所
引用: 1. Sørensen, H.P. and K.K. Mortensen, Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology, 2005. 115(2): p. 113-128. 2. Minuth, T., et al., Pilot scale processing of detergent-based aqueous two-phase systems. Biotechnology and Bioengineering, 1997. 55(2): p. 339-347. 3. Mateo, C., et al., Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. J Chromatogr A, 2001. 915(1-2): p. 97-106. 4. Nilsson, B. and L. Abrahmsén, [13] Fusions to staphylococcal protein A, in Methods in Enzymology, V.G. David, Editor 1990, Academic Press. p. 144-161. 5. Sakhamuru, K., D.W. Hough, and J.B. Chaudhuri, Protein purification by ultrafiltration using a beta-galactosidase fusion tag. Biotechnol Prog, 2000. 16(2): p. 296-8. 6. Schein, C.H., Production of Soluble Recombinant Proteins in Bacteria. Nat Biotech, 1989. 7(11): p. 1141-1149. 7. Smith, D.B. and K.S. Johnson, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 1988. 67(1): p. 31-40. 8. Terpe, K., Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol, 2003. 60(5): p. 523-33. 9. Knight, P., Downstream Processing. Nat Biotech, 1989. 7(8): p. 777-782. 10. 吳俊彥, 建立以冰核蛋白結合內含子之表面表現系統並應用於EGFP之生產2012: 中興大學化學工程研究所博士論文. 11. Samuelson, P., et al., Display of proteins on bacteria. Journal of Biotechnology, 2002. 96(2): p. 129-154. 12. 洪水根 and 汪德耀, 膜分子生物學1997, 基隆市: 水產出版社. 13. Benhar, I., Biotechnological applications of phage and cell display. Biotechnol Adv, 2001. 19(1): p. 1-33. 14. Freudl, R., et al., Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. J Mol Biol, 1986. 188(3): p. 491-4. 15. Charbit, A., et al., Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface. EMBO J, 1986. 5(11): p. 3029-37. 16. Stahl, S. and M. Uhlen, Bacterial surface display: trends and progress. Trends Biotechnol, 1997. 15(5): p. 185-92. 17. Wernérus, H. and S. Ståhl, Biotechnological applications for surface-engineered bacteria. 2004. 40(Pt 3): p. 209-228. 18. Wernerus, H. and S. Stahl, Biotechnological applications for surface engineered bacteria. Biotechnology and Applied Biochemistry, 2004. 40: p. 209-228. 19. Lee, S.Y., J.H. Choi, and Z. Xu, Microbial cell-surface display. Trends in Biotechnology, 2003. 21: p. 45-52. 20. Li, L., Kang, D. G., Cha, H. J., Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein. Biotechnology and Bioengineering 2004. 85: p. 214-221. 21. Wolber, P., Bacterial ice nucleation. Advances In Microbial Physiology 1993. 34: p. 203-237. 22. Zhao, J.L. and C.S. Orser, Conserved repetition in the ice nucleation gene inaX from Xanthomonas campestris pv. translucens. Molecular & general genetics, 1990. 223(1): p. 163-166. 23. Margaritis, A. and A.S. Bassi, Principles and biotechnological applications of bacterial ice nucleation. Critical Reviews in Biotechnology, 1991. 11: p. 277-295. 24. Edwards, A.R., et al., Unusual pattern of bacterial ice nucleation gene evolution. Molecular Biology and Evolution, 1994. 11: p. 911-920. 25. Kozloff, L., M. Turner, and F. Arellano, Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes. Journal of Bacteriology, 1991. 173: p. 6528-6536. 26. Kawahara, H., The structures and functions of ice crystal-controlling proteins from bacteria. Journal of Bioscience and Bioengineering, 2002. 94: p. 492-496. 27. Samuelson, P., et al., Display of proteins on bacteria. Journal of Biotechnology, 2002. 96: p. 129-154. 28. Jung, H.C., et al., Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein. Enzyme and Microbial Technology, 1998. 22: p. 348-354. 29. Wolber P and W. G, Bacterial ice-nucleation proteins. Trends in Biochemical Sciences, 1989. 14: p. 179-182. 30. Wolber, P.K., Bacterial ice nucleation. Advances in Microbial Physiology, 1993. 34: p. 203-237. 31. Jung, H.C., J.M. Lebeault, and J.G. Pan, Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nature Biotechnology, 1998. 16: p. 576-580. 32. Kim, Y.S., H.C. Jung, and J.G. Pan, Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants. Applied and Environmental Microbiology, 2000. 66: p. 788-793. 33. Shimazu, M., A. Mulchandani, and W. Chen, Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnology progress, 2001. 17: p. 76-80. 34. Shimazu, M., et al., Cell Surface Display of Organophosphorus Hydrolase in Pseudomonasputida Using an Ice Nucleation Protein Anchor. Biotechnology progress, 2003. 19(5): p. 1612-1614. 35. Wang, A.A., A. Mulchandani, and W. Chen, Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase. Applied and Environmental Microbiology, 2002. 68: p. 1684-1689. 36. Bae, W., A. Mulchandani, and W. Chen, Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. Journal of Inorganic Biochemistry, 2002. 88: p. 223-227. 37. Bassi, A.S., et al., Expression of Single Chain Antibodies (ScFvs) for c myc Oncoprotein in Recombinant Escherichiacoli Membranes by Using the Ice Nucleation Protein of Pseudomonassyringae. Biotechnology progress, 2000. 16: p. 557-563. 38. Kwak, Y.D., S.K. Yoo, and E.J. Kim, Cell surface display of human immunodeficiency virus type 1 gp120 on Escherichia coli by using ice nucleation protein. Clinical and Vaccine Immunology, 1999. 6: p. 499-503. 39. Lee, J.S., et al., Surface-displayed viral antigens on Salmonella carrier vaccine. Nature Biotechnology, 2000. 18: p. 645-648. 40. Van Bloois, E., et al., Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology, 2010. 41. Van Bloois, E., et al., Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis. Applied Microbiology and Biotechnology, 2009. 83: p. 679-687. 42. Wu, M.L., C.Y. Tsai, and T.H. Chen, Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity. FEMS Microbiology Letters, 2006. 256: p. 119-125. 43. Li, Q., et al., Improved phosphate biosorption by bacterial surface display of phosphate binding protein utilizing ice nucleation protein. FEMS Microbiology Letters, 2009. 299: p. 44-52. 44. Yang, C., et al., Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity. Applied and Environmental Microbiology, 2008. 74: p. 7733-7739. 45. Yang, C., et al., Cotranslocation of methyl parathion hydrolase to the periplasm and of organophosphorus hydrolase to the cell surface of Escherichia coli by the Tat pathway and ice nucleation protein display system. Applied and Environmental Microbiology, 2010. 76: p. 434-440. 46. Wu, P.H., R. Giridhar, and W.T. Wu, Surface display of transglucosidase on Escherichia coli by using the ice nucleation protein of Xanthomonas campestris and its application in glucosylation of hydroquinone. Biotechnology and Bioengineering, 2006. 95: p. 1138-1147. 47. Paulus, H., Inteins as enzymes. Bioorganic Chemistry, 2001. 29: p. 119-129. 48. Li SL, Zheng B, and Z. XM., Inteins and its biological significance. Biotechnology Letters, 2005. 16: p. 552-555. 49. Xie J, Huang J F, and L.C. Q, Analysis of the characteristic sequence of intein and revision of its motifs. Chinese Science Bulletin 2000. 45: p. 2525-2530. 50. Sun, Z., et al., Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli. Protein Expression and Purification, 2005. 43: p. 26-32. 51. Ding, Y., et al., Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing. Journal of Biological Chemistry, 2003. 278: p. 39133-39142. 52. Sun, P., et al., Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing. Journal of Molecular Biology, 2005. 353: p. 1093-1105. 53. Wu, H., M.Q. Xu, and X.Q. Liu, Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein. Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology, 1998. 1387: p. 422-432. 54. Williams, N.K., et al., In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein. Journal of Biological Chemistry, 2002. 277: p. 7790-7798. 55. Xu, M., H. Paulus, and S. Chong, Fusions to self-splicing inteins for protein purification. Methods in Enzymology, 2000. 326: p. 376-418. 56. Hao, D., K. Qian, and G. Shen, Protein splicing and its application in protein engineering. Yi chuan= Hereditas/Zhongguo yi chuan xue hui bian ji, 2004. 26: p. 249-252. 57. Xu, M.Q. and T.C. Evans, Intein-mediated ligation and cyclization of expressed proteins. Methods, 2001. 24: p. 257-277. 58. Shimomura, O., F. H. Johnson and Y. Saiga, Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol, 1962. 59: p. 223-239. 59. Brillet, K., et al., Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells. Cytotechnology, 2008. May;57(1): p. 101-109. 60. Halfie, M., GREEN FLUORESCENT PROTEIN. Photochemistry and Photobiology, 1995. 62(4): p. 651-656. 61. Kobayashi, T., et al., Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research. PLoS ONE., 2008. 3(12). 62. Roberto, F.F., J.M. Barnes, and D.F. Bruhn, Evaluation of a GFP reporter gene construct for environmental arsenic detection. Talanta, 2002. Aug 16;58(1): p. 181-188. 63. Tak, Y.K., et al., Green fluorescent protein (GFP) as a direct biosensor for mutation detection: elimination of false-negative errors in target gene expression. Analytical biochemistry, 2008. Sep 1;380(1): p. 91-98. 64. Travers, M.A., et al., Construction of a stable GFP-tagged Vibrio harveyi strain for bacterial dynamics analysis of abalone infection. FEMS Microbiology Letters, 2008. Dec;289(1): p. 34-40. 65. Wang, Z., et al., A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles. . Lab Chip., 2008. Nov;8(11): p. 1957-1962. 66. Zhang, G., V. Gurtu, and S.R. Kain, An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochemical and Biophysical Research Communications, 1996. 227(3): p. 707-711. 67. Morrison, D.A., Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol, 1979. 68: p. 326-331. 68. Hanahan, D., Studies on transformation of E.coli with plasmids. Journal of Molecular Biology, 1983. 166: p. 557-580. 69. Sambrook, J., E.F. Fritsch, and T. Maniastis, Molecular cloning . Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1989. 70. Banki, M.R., T.U. Gerngross, and D.W. Wood, Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Science, 2005. 14(6): p. 1387-1395. 71. Zhou, X., et al., Production of Recombinant Porcine Interferon alpha Using PHB–Intein-Mediated Protein Purification Strategy. Applied Biochemistry and Biotechnology, 2011. 163(8): p. 981-993. 72. Zhao, Z., et al., Purification of green fluorescent protein using a two-intein system. Applied Microbiology and Biotechnology, 2008. 77(5): p. 1175-1180. 73. Sun, Z., et al., Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli. Protein Expression and Purification, 2005. 43(1): p. 26-32. 74. Zhang, H.-Y., et al., Separation and purification of Escherichia coli-expressed human thymosin-[alpha]1 using affinity chromatography and high-performance liquid chromatography. Protein Expression and Purification, 2011. 77(2): p. 140-145. 75. McRae, S.R., C.L. Brown, and G.R. Bushell, Rapid purification of EGFP, EYFP, and ECFP with high yield and purity. Protein Expression and Purification, 2005. 41(1): p. 121-127.
摘要: 
前人所開發的細胞表面表現系統可直接生產胞外目標蛋白質,利用經修飾後之膜蛋白「冰核蛋白」(ice nucleation protein, INP) ,接上Intein (INT)斷裂蛋白質與目標蛋白重組成融合蛋白,此蛋白可以表現在細胞表面,並且利用Intein可進行自我剪切之特性,使得目標蛋白可從融合蛋白中分離而溶解在反應液中。
本研究主要探討此膜上表現系統於三種宿主細胞,ER2566、DH1、BL21(LPS free)中生產EGFP的差異,一併比較在不同pH值、不同的融合蛋白誘導時間以及不同斷裂時間下對EGFP產量以及純度的影響。
實驗結果顯示在相同的條件下,誘導時間在24小時EGFP產量以及純度表現比9小時佳。三種不同宿主細胞以大腸桿菌株ER2566表現最佳,在菌體經24小時誘導後,於pH6之條件下反應4小時可得EGFP純度62.5%;於pH7之條件下反應24小時可得EGFP最高產量82.3 mg/L。而在反應液 pH6與pH7之條件下,EGFP產物之產量與純度差異皆不顯著;然於pH7之條件下其產量較高,而於pH6之條件下其純度較高。
將原本系統中 intein 尾端所加之GRA序列片段移除後,其EGFP之產量降為原本之1/3倍,且純度亦降低,顯示GRA序列片段對於目標蛋白質之生產具有正面助益。

The cell surface expression system was constructed to produce extracellular enzyme in E. coli directly. The genes of the truncated ice nucleation protein (INP) along with intein (INT) and target protein, i.e., enchanced green fiuorescence protein (EGFP), were fused together to construct an INP-INT-EGFP gene, which was able to produce protein anchoring on cell membrane surface.
The performance of the production for EGFP with three different host cells, E. coli ER2566, DH1, BL21 (LPS free) was studied in surface expression system. The effects of different induction time, cleavage time and pH values of cleavage buffer on the yield and purity for EGFP were discussed.
From the experiments, it shows that under the same conditions, better performance was obtained for the yield and purity of EGFP under 24 h induction. Among the three different host cells, E. coli strain ER2566 had a better performance than the others. After 24 h induction with the cleavage condition of pH6 for 4 h, 62.5% purity can be obtained. The production of EGFP reached 82.3 mg/L under the cleavage condition of pH7 for 24 h. A higher yield of EGFP was achieved under pH7, whereas a higher purity was achieved under pH6.
The effect of intein segment with or without the additional GRA fragment on EGFP production was also compared. The result shows that with the GRA fragment in the terminal of intein, the yield and purity of EGFP production were both higher than that without GRA fragment. This indicates that the fragment of GRA has a positive effect on the production of the target protein.
URI: http://hdl.handle.net/11455/3154
其他識別: U0005-2108201213522900
Appears in Collections:化學工程學系所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.