Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3930
標題: 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
Production of recombinant enhanced green fluorescence protein via surface display system of ice nucleation protein coupling with self-cleavage intein
作者: 吳俊彥
Wu, Jiun-Yan
關鍵字: Protein production and purification;細胞表面表現;Surface display;Ice nucleation protein;Intein;Enhanced green fluorescent protein;Centrifugation;冰核蛋白;內含子;增強型綠螢光蛋白;重組蛋白;融合蛋白
出版社: 化學工程學系所
引用: 巫文玲,陳中成,張天鴻。1989。遺傳工程蛋白用藥市場調查。1 ed。財團法人生物技術開發中心,台北市。 洪水根,汪德耀。1997。膜分子生物學。水產出版社,基隆市。 Faber, K. 2000. Biotransformations in organic chemistry. 4 ed. Springer-Verlag, Berlin. Magnusson, S., Petersen, T.E., Sottrup-Jensen, L., Claey, H. 1975. Protease and Biological Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Roberts, S.M., Turner, N.J., Willetts, A.J., Turner, M.K. 1995. Introduction to biocatalysis using enzymes and micro-organisms. Cambridge University Press, Cambridge. Andersen, D.C., Krummen, L. 2002. Recombinant protein expression for therapeutic applications. Current Opinion in Biotechnology, 13(2), 117-123. Arnau, J., Lauritzen, C., Petersen, G.E., Pedersen, J. 2006. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein expression and purification, 48(1), 1-13. Banki, M.R., Gerngross, T.U., Wood, D.W. 2005. Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Science, 14(6), 1387-1395. Banki, M.R., Wood, D.W. 2005. Inteins and affinity resin substitutes for protein purification and scale up. Microbial Cell Factories, 4, 32. Benhar, I. 2001. Biotechnological applications of phage and cell display. Biotechnology Advances, 19(1), 1-33. Bingle, W.H., Nomellini, J.F., Smit, J. 1997. Cell-surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S-layer of Caulobacter crescentus. Molecular Microbiology 26, 277-288. 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, 248-254. Breinig, F., Schmitt, M.J. 2002. Spacer-elongated cell wall fusion proteins improve cell surface expression in the yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 58(5), 637-644. Brillet, K., Perret, B.G., Klein, V., Pattus, F., Wagner, R. 2008. Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells. Cytotechnology, May;57(1), 101-109. Cantor, E.J., Chong, S. 2001. Intein-mediated rapid purification of Cre recombinase. Protein expression and purification, 22(1), 135-140. Charbit, A., Boulain, J.C., Ryter, A., Hofnung, M. 1986. Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface. The EMBO journal, 5(11), 3029-3037. Chiang, C.J., Chen, H.C., Chao, Y.P., Tzen, J.T. 2005. Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli. Journal of Agricultural and Food Chemistry, 53(12), 4799-4804. Derbyshire, V., Wood, D.W., Wu, W., Dansereau, J.T., Dalgaard, J.Z., Belfort, M. 1997. Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution. Proceedings of the National Academy of Sciences of the United States of America, 94(21), 11466-11471. Esipov, R.S., Stepanenko, V.N., Chupova, L.A., Boyarskikh, U.A., Filipenko, M.L., Miroshnikov, A.I. 2008. Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system. Protein expression and purification, Sep;61(1), 1-6. Felix, H. 1982. Permeabilized cells. Analytical Biochemistry, 120(2), 211-234. Fischer, B., Sumner, I., Goodenough, P. 1993. Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnology and Bioengineering, 41(1), 3-13. Fong, B.A., Wu, W.-Y., Wood, D.W. 2010. The potential role of self-cleaving purification tags in commercial-scale processes. Trends in Biotechnology, 28(5), 272-279. Francisco, J.A., Earhart, C. F., Georgiou, G. 1992. Transport and anchoring of blactamase to the external surface of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 89, 2713-2717. Freudl, R., MacIntyre, S., Degen, M., Henning, U. 1986. Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. Journal of molecular biology, 188(3), 491-494. Gogarten, J.P., Senejani, A.G., Zhaxybayeva, O., Olendzenski, L., Hilario, E. 2002. INTEINS: Structure, Function, and Evolution. Annual Review of Microbiology, 56(1), 263-287. Guo, C., Li, Z., Shi, Y., Xu, M., Wise, J.G., Trommer, W.E., Yuan, J. 2004. Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin. Protein expression and purification, 37(2), 361-367. Halfie, M. 1995. GREEN FLUORESCENT PROTEIN. Photochemistry and Photobiology, 62(4), 651-656. Hearn, M.T., Acosta, D. 2001. Applications of novel affinity cassette methods: use of peptide fusion handles for the purification of recombinant proteins. Journal of Molecular Recognition, 14(6), 323 - 369. Hong, J., Wang, Y., Ye, X., Zhang, Y.H.P. 2008. Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. Journal of Chromatography A, 1194(2), 150-154. Jung, H.C., H., P.J., S.H., P., J.M., L., J.G., P. 1998a. Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein. Enzyme and Microbial Technology, 22, 348-354. Jung, H.C., Lebeault, J.M., Pan, J.G. 1998b. Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nature Biotechnology, 16(6), 576-580. Kim, E.J., Yoo, S.K. 1999. Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein. Letters in Applied Microbiology, 29(5), 292-297. Klauser, T., Pohlner, J., Meyer, T.F. 1990. Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation. The EMBO journal, 22, 348-354. Knight, P. 1989. Downstream Processing. Nature Biotechnology, 7, 777-782. Kobayashi, T., Morone, N., Kashiyama, T., Oyamada, H., Kurebayashi, N., Murayama, T. 2008. Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research. PLoS ONE, 3(12). Kondo, A., Ueda, M. 2004. Yeast cell-surface display—applications of molecular display. Applied Microbiology and Biotechnology, 64(1), 28-40. Laemmli, U.K. 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227(5259), 680-685. Lee, S.F., March, R.J., Halperin, S.A., Faulkner, G., Gao, L. 1999. Surface expression of a protective recombinant pertussis toxin S1 subunit fragment in Streptococcus gordonii. Infection and Immunity, 67, 1511-1516. Lee, S.H., Choi, J.I., Han, M.J., Choi, J.H., Lee, S.Y. 2005. Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent. Biotechnol Bioeng, 90, 223-230. Lee, S.Y., Choi, J.H., Xu, Z. 2003. Microbial cell-surface display. Trends in Biotechnology, 21(1), 45-52. Li, L., Kang, D. G., Cha, H. J. 2004. Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein. Biotechnology and Bioengineering, 85, 214-221. Liu, X.-Q. 2000. PROTEIN-SPLICING INTEIN: Genetic Mobility, Origin, And Evolution. Annual Review of Genetics, 34(1), 61-76. Maity, S., Animesh, K.D., Chattopadhyay, A. 2009. Seed protein polymorphism in nine species of Jute (Corchorus, Family: Tiliaceae). Indian Journal of Science and Technology, 2, 34-36. Mateo, C., Fernandez-Lorente, G., Pessela, B.C., Vian, A., Carrascosa, A.V., Garcia, J.L., Fernandez-Lafuente, R., Guisan, J.M. 2001. 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, 915, 97-106. Mathys, S., EvansJr, T.C., Chute, I.C., Wu, H., Chong, S., Benner, J., Liu, X.-Q., Xu, M.-Q. 1999. Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene, 231(1-2), 1-13. McRae, S.R., Brown, C.L., Bushell, G.R. 2005. Rapid purification of EGFP, EYFP, and ECFP with high yield and purity. Protein Expression and Purification, 41(1), 121-127. Minuth, T., Gieren, H., Pape, U., Raths, H.C., Thömes, J., Kula, M.R. 1997. Pilot scale processing of detergent-based aqueous two-phase systems. Biotechnology and Bioengineering, 55(2), 339-347. Neu, H.C., Heppel, L.A. 1965. The Release of Enzymes from Escherichia coli by Osmotic Shock and during the Formation of Spheroplasts. Journal of Biological Chemistry, 240(9), 3685-3692. Nilsson, B., Abrahmsén, L. 1990. Fusions to staphylococcal protein A. Methods in Enzymology, 185, 144-161. Nossal, N.G., Heppel, L.A. 1966. The Release of Enzymes by Osmotic Shock from Escherichia coli in Exponential Phase. Journal of Biological Chemistry, 241(13), 3055-3062. Parks, T.D., Leuther, K.K., Howard, E.D., Johnston, S.A., Dougherty, W.G. 1994. Release of Proteins and Peptides from Fusion Proteins Using a Recombinant Plant Virus Proteinase. Analytical Biochemistry, 216(2), 413-417. Paulus, H. 2001. Inteins as enzymes. Bioorganic Chemistry, 29(3), 119-129. Peng, C.C., Chen, J.C., Shyu, D.J., Chen, M.J., Tzen, J.T. 2004. A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides. Journal of biotechnology, 111(1), 51-57. Perler, F.B. 2002a. InBase: the Intein Database. Nucleic Acids Research, 30(1), 383-384. Perler, F.B., Davis, E.O., Dean, G.E., Gimble, F.S., Jack, W.E., Neff, N., Noren, C.J., Thorner, J., Belfort, M. 1994. Protein splicing elements: inteins and exteins -- a definition of terms and recommended nomenclature. Nucleic Acids Research, 22(7), 1125-1127. Perler, F.B., Xu, M.-Q., Paulus, H. 1997. Protein splicing and autoproteolysis mechanisms. Current Opinion in Chemical Biology, 1(3), 292-299. Pietrokovski, S. 1994. Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins. Protein Science, 3(12), 2340-2350. Pietrokovski, S. 2001. Intein spread and extinction in evolution. Trends in Genetics, 17(8), 465-472. Rathsam, C., Giffard, P.M., Jacques, N.A. 1993. The cell bound fructosyltransferase of Streptococcus sali arius: the carboxyl terminus specifies attachment in a Streptococcus gordonii model system. The Journal of Bacteriology, 175, 4520-4527. Roberto, F.F., Barnes, J.M., Bruhn, D.F. 2002. Evaluation of a GFP reporter gene construct for environmental arsenic detection. Talanta, Aug 16;58(1), 181-188. Sakhamuru, K., Hough, D.W., Chaudhuri, J.B. 2000. Protein Purification by Ultrafiltration Using a beta-Galactosidase Fusion Tag. Biotechnology progress, 16(2), 296-298. Samuelson, P., Gunneriusson, E., Nygren, P.A., Stahl, S. 2002. Display of proteins on bacteria. Journal of biotechnology, 96(2), 129-154. Schein, C.H. 1989. Production of Soluble Recombinant Proteins in Bacteria. Nature Biotechnology, 7(11), 1141-1149. Schorr, J., Knapp, B., Hundt, E., Kupper, H.A., Amann, E. 1991. Surface expression of malarial antigens in Salmonella typhimurium: induction of serum antibody response upon oral vaccination of mice. Vaccine, 9, 675-681. Sharma, S., Zhang, A., Wang, H., Harcum, S.W., Chong, S. 2003. Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions. Biotechnology progress, 19(3), 1085-1090. Shemella, P., Pereira, B., Zhang, Y., Van Roey, P., Belfort, G., Garde, S., Nayak, S.K. 2007. Mechanism for Intein C-Terminal Cleavage: A Proposal from Quantum Mechanical Calculations. Biophysical Journal, 92(3), 847-853. Shimazu, M., Mulchandani, A., Chen, W. 2001. Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnology Progress, 17(1), 76-80. Shimazu, M., Nguyen, A., Mulchandani, A., Chen, W. 2003. Cell surface display of organophosphorus hydrolase in Pseudomonas putida using an ice-nucleation protein anchor. Biotechnology Progress, 19(5), 1612-1614. Shimomura, O., F. H. Johnson and Y. Saiga. 1962. Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Journal of Cellular and Comparative Physiology, 59, 223-239. Singleton, S.F., Simonette, R.A., Sharma, N.C., Roca, A.I. 2002. Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein. Protein expression and purification, 26(3), 476-88. Smith, D.B., Johnson, K.S. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 67(1), 31-40. Smith, G.P. 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 228(4705), 1315-1317. Ståhl, S., Uhlén, M. 1997. Bacterial surface display: trends and progress. Trends in Biotechnology, 15(5), 185-192. Stoddard, B.L. 2005. Homing endonuclease structure and function. Quarterly Reviews of Biophysics, 38(1), 49-95. Stover, C.K., Bansal, G.P., Langerman, S., Hanson, M.S. 1994. Protective immunity elicited by rBCG vaccines. Developments in biological standardization, 82, 163-170. Sun, Z., Chen, J., Yao, H., Liu, L., Wang, J., Zhang, J., Liu, J.-N. 2005a. 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, 43(1), 26-32. Szweda, P., Pladzyk, R., Kotlowski, R., Kur, J. 2001. Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system. Protein expression and purification, 22(3), 467-471. Tak, Y.K., Naoghare, P.K., Lee, K.H., Park, S.S., Song, J.M. 2008. Green fluorescent protein (GFP) as a direct biosensor for mutation detection: elimination of false-negative errors in target gene expression. Analytical biochemistry, 380(1), 91-98. Terpe, K. 2003. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60(5), 523-533. Thapa, A., Shahnawaz, M., Karki, P., Dahal, G.R., Sharoar, M.G., Shin, S.Y., Lee, J.S., Cho, B., Park, I.-S. 2008. Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins. BioTechniques, 44, 787-796. Titchener-Hooker, N.J., Gritsis, D., Olbrich, R., Gardiner, S.A.M., Mannweiler, K., Fish, N.M., Hoare, M. 1991. Integrated Process Design for Producing and Recovering Proteins from Inclusion Bodies. Biopharm International, 4(7), 34-38. Travers, M.A., Barbou, A., Le Goïc, N., Huchette, S., Paillard, C., M., K. 2008. Construction of a stable GFP-tagged Vibrio harveyi strain for bacterial dynamics analysis of abalone infection. FEMS microbiology letters, 289(1), 34-40. Wang, A.A., Mulchandani, A., Chen, W. 2002. 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, 68(4), 1684-1689. Wang, Z., Wu, H., Chen, J., Zhang, J., Yao, Y., Chen, G.Q. 2008. A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles. Lab on a Chip, 8(11), 1957-1962. Wernérus, H., Ståhl, S. 2004. Biotechnological applications for surface-engineered bacteria. Biotechnology and applied biochemistry, 40(3), 209-228. Wolber, P. 1993. Bacterial ice nucleation. Advances In Microbial Physiology, 34, 203-237. Wood, D.W., Derbyshire, V., Wu, W., Chartrain, M., Belfort, M., Belfort, G. 2000. Optimized Single-Step Affinity Purification with a Self-Cleaving Intein Applied to Human Acidic Fibroblast Growth Factor. Biotechnology Progress, 16(6), 1055-1063. Wu, P.H., Giridhar, R., Wu, W.T. 2006. 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, 95(6), 1138-1147. Xu, M.Q., Evans, T.C., Jr. 2001. Intein-mediated ligation and cyclization of expressed proteins. Methods, 24(3), 257-277. Xu, M.Q., Paulus, H., Chong, S. 2000. Fusions to self-splicing inteins for protein purification. Methods in Enzymology, 326, 376-418. Zhang, A., Gonzalez, S.M., Cantor, E.J., Chong, S. 2001. Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins. Gene, 275(2), 241-252. Zhang, G., Brokx, S., Weiner, J.H. 2005. Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature Biotechnology, 24, 100-104. Zhang, G., Gurtu, V., Kain, S.R. 1996. An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochemical and Biophysical Research Communications, 227(3), 707-711. Zhang, H.-Y., Chen, P.-F., Xu, J.-M., Dai, Q.-M., Xu, F., Han, Q.-W., Wang, J.-J., Jin, H.-Y. 2011. Separation and purification of Escherichia coli-expressed human thymosin-[alpha]1 using affinity chromatography and high-performance liquid chromatography. Protein Expression and Purification, 77(2), 140-145. Zhao, J.L., Orser, C.S. 1990. Conserved repetition in the ice nucleation gene inaX from Xanthomonas campestris pv. translucens. Molecular and General Genetics MGG, 223(1), 163-166. Zhao, Z., Lu, W., Dun, B., Jin, D., Ping, S., Zhang, W., Chen, M., Xu, M.-Q., Lin, M. 2008. Purification of green fluorescent protein using a two-intein system. Applied Microbiology and Biotechnology, 77(5), 1175-1180. Zhou, X., Song, Z., Liu, X., Jia, F., Wang, Y. 2011. Production of Recombinant Porcine Interferon alpha Using PHB–Intein-Mediated Protein Purification Strategy. Applied Biochemistry and Biotechnology, 163(8), 981-993.
摘要: 
重組蛋白質純化方式操作複雜,設備成本高,不利於大規模生產以供學術及工業使用。本研究構築一細胞表面表現系統,以基因工程技術將冰核蛋白(ice nucleation protein, INP) 與內含子(intein, INT) 重組成融合蛋白INP-INT,並利用增強型綠螢光蛋白(enhanced green fluorescence protein, EGFP)為模型蛋白,建構一個具量產及純化重組蛋白質之製程模型。此表面表現系統可以將目標蛋白表現在細胞表面,藉intein之自我裂解反應可以將目標蛋白EGFP釋放,而達到生產並純化EGFP之目的。利用本系統進行生產EGFP,不需要借助昂貴的設備器材,也不需要複雜的步驟程序,僅僅利用幾次離心即可由上層液中獲得產物。
本研究選殖pTWIN1上之 Ssp DnaB intein基因,並將其C端連接選殖自pEGFP上之EGFP片段,形成INT-EGFP之基因片段後,再將此片段送入取自成功大學吳文騰教授實驗室建構之pInaXNC-aglA2中,並連結於其上經修飾之冰核蛋白之C端,得到質體pINP-INT-EGFP。以E. coli DH1做為INP-INT-EGFP融合蛋白表現之宿主細胞,經IPTG誘導後,可得到大小約為74kDa之INP-INT-EGFP融合蛋白。
重組E. coli細胞經誘導生產融合蛋白後,置於反應液中進行intein斷裂反應,結果顯示在高溫、高pH值及低鹽度之條件下可得到較高之EGFP產量。經誘導後之菌體直接置於37℃、pH 11及0.1% (w/v) NaCl之反應液下進行反應,可於第五天得EGFP最高產量278 mg/L,其純度約為50%。
鬆散細胞膜結構可以促進intein於緩和之條件下進行斷裂反應。於pH 8之反應液中添入0.5% (v/v)之界面活性劑(Triton X-100),於37℃下進行intein斷裂反應後,可於第一天得到EGFP產物184 mg/L,回收率為85%,但其純度僅27%。菌體先以0.5% (v/v)之界面活性劑(Triton X-100)處理一小時後,再置換於乾淨反應液中進行intein斷裂反應一天,則可提升EGFP純度至65%,但其產物僅可得40 mg/L,回收率為19%。若以滲透壓衝擊法先破壞細胞膜結構,將間質蛋白先洗出後,可以提高產物純度。菌體經高低張溶液處理後,再置換pH 8之反應液中以37℃進行intein斷裂反應,可於第一天得到EGFP產物198 mg/L,回收率為91%,其純度可提升至60%。
而經誘導後之菌體以壓力式破菌機於15kpsi之壓力下進行破碎後,將所回收之不可溶細胞破片置於反應溫度37℃,pH 8之反應液中,經反應一天可以獲得EGFP 產量63 mg/L,其純度可高達97%,回收率29%。
使用本系統生產純化蛋白質無需進行額外之蛋白質分離純化程序,僅利用離心程序即可得到高純度之蛋白質產物。若能繼續改良此量產蛋白質並純化之醱酵工程技術平台,不但可以降低蛋白質純化成本,還可以進一步取得大量之高純度目標蛋白,以應用於醫療、學術研究以及工業發展上。

Protein production and purification is often costly and laborious due to the complicated processes. It is highly expected to develop a simple process to obtain high purity protein without the complicate purification steps and costly equipments. In this study, a novel recombinant protein production system was developed by using cell surface display segment to finally produce a model protein entity. The system is constructing a fusion protein with a self-cleavage intein along with a cell membrane motife, ice nucleation protein, and a model protein, enhanced green fluorescence protein (EGFP). By using this system, the target protein (here is EGFP in this case) can be separated from the cell membrane by the auto-proteolysis property of intein. The protein production can be obtained in the supernatant by only several centrifugations, no complicate processes or expensive equipments are required. .
By simply holding the cell pellets in Tris-HCl (pH 11) with 0.1% (w/v) NaCl buffer at 37℃, EGFP was solubilized from the INP-INT segment embedded on the cell surface via intein's self-cleavage function. The EGFP concentration of 278 mg/L with the purity of 50% was obtained at day 5. The EGFP can be harvested only via centrifugation, and no cell disruption process is required.
Cell membrane damage is helpful for EGFP release in neutral condition. By adding 0.5% Triton X-100 in the cleavage buffer (20 mM Tris–HCl, 1 mM EDTA and 1% NaCl) of pH 8, the EGFP was obtained 184 mg/L with the purity of 27% after reaction for 1 day, and the recovery was 85%. Purity of EGFP production can be increased to 65% by replacing cell pellet into clean cleavage buffer (pH 8) after Triton X-100 pretreatment for 1h, and EGFP production was 40 mg/L with the recovery of 19%. Cell membrane can also be damaged by osmotic shock. In the process of osmotic shock, cells were treated with a concentrated solution of sucrose and then shifted to water, and the impurities in cell periplasm were washed out by the water, followed by shifting the cells into the cleavage buffer (pH 8), EGFP production was 198 mg/L with the recovery of 91% and the purity of 60%. On the other hand, when applying cell disruption in the process, EGFP of 63 mg/L with the purity of 97% and the recovery of 29% was obtained.
This study discloses a facile approach producing recombinant protein. Due to the simplicity of the approach, the protein production and purification can be simultaneously carried out in few steps, which might help its realiation in the scale up process. This approach is expected to be an alternative way for recombinant protein production in bio-industry for academic and industrial use.
URI: http://hdl.handle.net/11455/3930
其他識別: U0005-3001201219310100
Appears in Collections:化學工程學系所

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