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標題: 建立INP-INT膜上表現系統並應用於D-Hydantoinase 生產之研究
Development of the INP-INT expression system and its application on D-hydantoinase production
作者: 劉姿岑
Liu, Tzu-Tsen
關鍵字: ecombinant protein;重組蛋白質;ice nucleation protein;intein;hydantoinase;surface display;冰核蛋白;Intein;Hydantoinase;表面表現
出版社: 化學工程學系所
引用: 1. Sorensen, H.P. and Mortensen, K.K., Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology, 2005. 115: p. 113-128. 2. Mateo, C., Fernandez-Lorente, G., Pessela, B.C.C., Vian, A., Carrascosa, A.V., Garcia, J.L., Fernandez-Lafuente, R., and Guisan, J.M., Affinity chromatography of polyhistidine tagged enzymes:: New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. Journal of Chromatography A, 2001. 915: p. 97-106. 3. Nilsson, B. and Abrahmsen, L., Fusions to staphylococcal protein A. Methods in Enzymology, 1990. 185: p. 144-161. 4. Sakhamuru, K., Hough, D.W., and Chaudhuri, J.B., Protein Purification by Ultrafiltration Using a beta-Galactosidase Fusion Tag. Biotechnology progress, 2000. 16: p. 296-298. 5. Schein, C.H., Production of soluble recombinant proteins in bacteria. Nature Biotechnology, 1989. 7: p. 1141-1149. 6. Smith, D.B. and Johnson, K.S., Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 1988. 67: p. 31-40. 7. Terpe, K., Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 2003. 60: p. 523-533. 8. Thapa, A., Shahnawaz, M., Karki, P., Dahal, G.R., Sharoar, M.G., Shin, S.Y., Lee, J.S., Cho, B., and Park, I.S., Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins. BioTechniques, 2008. 44: p. 787-798. 9. Titchener-Hooker, N., Gritsis, D., Mannweiler, K., Olbrich, R., Gardiner, S., Fish, N., and Hoare, M., Integrated process design for producing and recovering proteins from inclusion bodies. Biopharm International 1991. 4: p. 34-38. 10. Fischer, B., Sumner, I., and Goodenough, P., Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnology and Bioengineering, 1993. 41: p. 3-13. 11. Knight, P., Downstream Processing. Nature Biotechnology, 1989. 7: p. 777-782. 12. Benhar, I., Biotechnological applications of phage and cell display. Biotechnology Advances, 2001. 19: p. 1-33. 13. Freudl, R., MacIntyre, S., Degen, M., and Henning, U., Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. Journal of Molecular Biology, 1986. 188: p. 491-494. 14. Charbit, A., Boulain, J., Ryter, A., and Hofnung, M., Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface. EMBO Journal, 1986. 5: p. 3029-3037. 15. Samuelson, P., Gunneriusson, E., Nygren, P.A., and Stahl, S., Display of proteins on bacteria. Journal of Biotechnology, 2002. 96: p. 129-154. 16. Stahl, S. and Uhlen, M., Bacterial surface display: trends and progress. Trends in Biotechnology, 1997. 15: p. 185-192. 17. Wernerus, H. and Stahl, S., Biotechnological applications for surface engineered bacteria. Biotechnology and Applied Biochemistry, 2004. 40: p. 209-228. 18. Lee, S.Y., Choi, J.H., and Xu, Z., Microbial cell-surface display. Trends in Biotechnology, 2003. 21: p. 45-52. 19. Margaritis, A. and Bassi, A.S., Principles and biotechnological applications of bacterial ice nucleation. Critical Reviews in Biotechnology, 1991. 11: p. 277-295. 20. Edwards, A.R., Van Den Bussche, R.A., Wichman, H.A., and Orser, C.S., Unusual pattern of bacterial ice nucleation gene evolution. Molecular Biology and Evolution, 1994. 11: p. 911-920. 21. Kozloff, L., Turner, M., and Arellano, F., Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes. Journal of Bacteriology, 1991. 173: p. 6528-6536. 22. Kawahara, H., The structures and functions of ice crystal-controlling proteins from bacteria. Journal of Bioscience and Bioengineering, 2002. 94: p. 492-496. 23. Jung, H.C., Park, J.H., Park, S.H., Lebeault, J.M., and Pan, J.G., Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein. Enzyme and Microbial Technology, 1998. 22: p. 348-354. 24. Wolber P and G, W., Bacterial ice-nucleation proteins. Trends in Biochemical Sciences, 1989. 14: p. 179-182. 25. Wolber, P.K., Bacterial ice nucleation. Advances in Microbial Physiology, 1993. 34: p. 203-237. 26. Jung, H.C., Lebeault, J.M., and Pan, J.G., Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nature Biotechnology, 1998. 16: p. 576-580. 27. Kim, Y.S., Jung, H.C., and Pan, J.G., Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants. Applied and Environmental Microbiology, 2000. 66: p. 788-793. 28. Shimazu, M., Mulchandani, A., and Chen, W., Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnology progress, 2001. 17: p. 76-80. 29. Shimazu, M., Nguyen, A., Mulchandani, A., and Chen, W., Cell Surface Display of Organophosphorus Hydrolase in Pseudomonasputida Using an Ice Nucleation Protein Anchor. Biotechnology progress, 2003. 19(5): p. 1612-1614. 30. Wang, A.A., Mulchandani, A., and Chen, W., 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. 31. Bae, W., Mulchandani, A., and Chen, W., Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. Journal of Inorganic Biochemistry, 2002. 88: p. 223-227. 32. Bassi, A.S., Ding, D.N., Gloor, G.B., and Margaritis, A., 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. 33. Kwak, Y.D., Yoo, S.K., and Kim, E.J., 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. 34. Lee, J.S., Shin, K.S., Pan, J.G., and Kim, C.J., Surface-displayed viral antigens on Salmonella carrier vaccine. Nature Biotechnology, 2000. 18: p. 645-648. 35. Van Bloois, E., Winter, R.T., Kolmar, H., and Fraaije, M.W., Decorating microbes: surface display of proteins on Escherichia coli. Trends in Biotechnology, 2010. 36. Van Bloois, E., Winter, R.T., Janssen, D.B., and Fraaije, M.W., 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. 37. Wu, M.L., Tsai, C.Y., and Chen, T.H., 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. 38. Li, Q., Yu, Z., Shao, X., He, J., and Li, L., Improved phosphate biosorption by bacterial surface display of phosphate binding protein utilizing ice nucleation protein. FEMS Microbiology Letters, 2009. 299: p. 44-52. 39. Yang, C., Zhu, Y., Yang, J., Liu, Z., Qiao, C., Mulchandani, A., and Chen, W., 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. 40. Yang, C., Freudl, R., Qiao, C., and Mulchandani, A., 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. 41. Wu, P.H., Giridhar, R., and Wu, W.T., 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. 42. Paulus, H., Inteins as enzymes. Bioorganic Chemistry, 2001. 29: p. 119-129. 43. Li SL, Zheng B, and XM., Z., Inteins and its biological significance. Biotechnology Letters, 2005. 16: p. 552-555. 44. Xie J, Huang J F, and Q, L.C., Analysis of the characteristic sequence of intein and revision of its motifs. Chinese Science Bulletin 2000. 45: p. 2525-2530. 45. Sun, Z., Chen, J., Yao, H., Liu, L., Wang, J., Zhang, J., and Liu, J.N., 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. 46. Ding, Y., Xu, M.Q., Ghosh, I., Chen, X., Ferrandon, S., Lesage, G., and Rao, Z., 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. 47. Sun, P., Ye, S., Ferrandon, S., Evans, T.C., Xu, M.Q., and Rao, Z., 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. 48. Wu, H., Xu, M.Q., and Liu, X.Q., 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. 49. Williams, N.K., Prosselkov, P., Liepinsh, E., Line, I., Sharipo, A., Littler, D.R., Curmi, P.M.G., Otting, G., and Dixon, N.E., In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein. Journal of Biological Chemistry, 2002. 277: p. 7790-7798. 50. Xu, M., Paulus, H., and Chong, S., Fusions to self-splicing inteins for protein purification. Methods in Enzymology, 2000. 326: p. 376-418. 51. Hao, D., Qian, K., and Shen, G., Protein splicing and its application in protein engineering. Yi chuan= Hereditas/Zhongguo yi chuan xue hui bian ji, 2004. 26: p. 249-252. 52. Xu, M.Q. and Evans, T.C., Intein-mediated ligation and cyclization of expressed proteins. Methods, 2001. 24: p. 257-277. 53. Louwrier, A. and Knowles, C.J., The aim of industrial enzymic amoxycillin production: characterization of a novelcarbamoylase enzyme in the form of a crude, cell free extract. Biotechnology and Applied Biochemistry, 1997. 25: p. 143-149. 54. Olivieri, R., Fascetti, E., Angelini, L., and Degen, L., Microbial transformation of racemic hydantoins to D amino acids. Biotechnology and Bioengineering, 1981. 23: p. 2173-2183. 55. Brooks, K.P., Jones, E.A., Kim, B.D., and Sander, E.G., Bovine liver dihydropyrimidine amidohydrolase: purification, properties, and characterization as a zinc metalloenzyme. Archives of Biochemistry and Biophysics, 1983. 226: p. 469-483. 56. Luksa, V., Starkuviene, V., Starkuviene, B., and Dagys, R., Purification and characterization of the D-hydantoinase from Bacillus circulans. Applied Biochemistry and Biotechnology, 1997. 62: p. 219-231. 57. Mukohara, Y., Ishikawa, T., Watabe, K., and Nakamura, H., A thermostable hydantoinase of Bacillus stearothermophilus NS1122A: cloning, sequencing, and high expression of the enzyme gene, and some properties of the expressed enzyme. Bioscience, Biotechnology, and Biochemistry, 1994. 58: p. 1621-1626. 58. SHIMIZU, S., SHIMADA, H., TAKAHASHI, S., OHASHI, T., TANI, Y., and YAMADA, H., Synthesis of N-Carbamyl-D-2-thienyl-glycine and D-2-Thienylglycine by Microbial Hydantoinase. Agricultural and Biological Chemistry, 1980. 44(9): p. 2233-2234. 59. Olivieri, R., Fascetti, E., Angelini, L., and Degen, L., Microbial transformation of racemic hydantoins to D amino acids. Biotechnology and Bioengineering, 1981. 23(10): p. 2173-2183. 60. Morin, A., Hummel, W., and Kula, M.R., Production of hydantoinase fromPseudomonas fluorescens strain DSM 84. Applied Microbiology and Biotechnology, 1986. 25: p. 91-96. 61. Runser, S. and Ohleyer, E., Properties of the hydantoinase from Agrobacterium sp. IP I-671. Biotechnology Letters, 1990. 12: p. 259-264. 62. Yamada, H., Takahashi, S., Kii, Y., and Kumagai, H., Distribution of Hydantoin Hydrolyzing Activity in Microorganisms: Microbial Transformation of Hydantoins to Amino Acids (I). Journal of Fermentation Technology, 1978. 56: p. 484-491. 63. Chao, Y.P., Juang, T.Y., Chern, J.T., and Lee, C.K., Production of Dp-Hydroxyphenylglycine by N-Carbamoyl-D-amino Acid Amidohydrolase-Overproducing Escherichia coli Strains. Biotechnology progress, 1999. 15: p. 603-607. 64. Morrison, D.A., Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol, 1979. 68: p. 326-331. 65. Hanahan, D., Studies on transformation of E.coli with plasmids. Journal of Molecular Biology, 1983. 166: p. 557-580. 66. Sambrook, J., Fritsch, E.F., and Maniastis, T., Molecular cloning . Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1989. 67. Wu, W.Y., Miller, K.D., Coolbaugh, M., and Wood, D.W., Intein-mediated one-step purification of E. coli secreted human antibody fragments. Protein Expression and Purification, 2010. 76: p. 221-228. 68. Kosinski, M.J., Rinas, U., and Bailey, J.E., Isopropyl-β-D-thiogalactopyranoside influences the metabolism of Escherichia coli. Applied Microbiology and Biotechnology, 1992. 36: p. 782-784. 69. Weickert, M.J., Doherty, D.H., Best, E.A., and Olins, P.O., Optimization of heterologous protein production in Escherichia coli. Current Opinion in Biotechnology, 1996. 7: p. 494-499.
生產重組蛋白質以往皆利用大腸桿菌可以短時間內快速生長的特性生產目標蛋白,但是純化蛋白質過程瑣碎耗時,並且蛋白質經大量表現後也有可能會產生沒有蛋白質活性(包涵體,inclusion body)的問題,增加生產的困擾。本研究運用細胞表面表現系統直接生產胞外目標酵素,利用經基因剪切修飾後之膜蛋白「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斷裂蛋白質對pH值敏感性,進行自我剪切,則D-Hydantoinase (Dht)會從融合蛋白中分離而溶解在Cleavage buffer中。重組蛋白誘導條件為:將含有表現Dht質體的E.coli 在37 ºC,200 rpm下培養,當菌體量達OD600為0.6時,加入IPTG 使其最後濃度為0.05mM,於15 ºC誘導表現24小時。收集經誘導後的菌體,再懸浮於Cleavage buffer (pH6)中,在25 ºC,100 rpm下進行斷裂反應。經HPLC分析後,在搖瓶發酵液中可獲得活性約0.225 U/ml的Dht酵素。利用TotalLab v2.01分析SDS- PAGE中可獲得48.7 %的純度。

The production of recombinant proteins usually employs E. coli due to its fast growth and easy manipulation. However, the purification of the protein is time-consuming and complicated. Meanwhile, over expression of the recombinant protein would result in inactive protein (i.e. inclusion body) and hinder the protein production.
In this study, the cell surface expression system was used to directly produce extracellular enzyme. The genes of the truncated ice nucleation protein (INP) along with intein (INT)and target protein, here is D-Hydantoinase (Dht), were fused together to construct an INP-INT-Dht gene, which was able to produce fusion protein on cell membrane surface.
The induction conditions for E. coli with the constructed plasmid to produce fusion protein are as follows: E.coli was incubated at 37 ºC and 200 rpm till OD600 reached 0.6. Then, IPTG was added to the broth and the induction was conducted at 15 ºC for 24 h. The cell was harvested and resuspended in the cleavage buffer of pH6, 25 ºC, and 100 rpm for 24 h. A centrifugation was performed to remove the cell from the buffer and the supernatant was collected. The harvested Dht with the activity of 0.225 U/ml and a purity of 48.7% was obtained.
其他識別: U0005-0908201118591600
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