Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3643
標題: 以固定化金屬吸附材進行蛋白質復性之研究
Application of immobilized metal affinity adsorbents for protein refolding
作者: 黃柏儒
Huang, Po-Ju
關鍵字: refolding
復性
inclusion body
內函體
出版社: 化學工程學系所
引用: 李晏忠。利用蛋白質之工程技術改造N-acetyl-D-glucosamine-2-epimerase及N-acetyl-neuraminate aldoase的性質,工業技術研究院委託學術機構研究報告,中興大學,台中。(1999) 陳淑芬,大腸桿菌中表現N-acetyl-D-glucosamine 2-epimerase基因,中興大學碩士論文(1999) 楊傑予,澱粉分解酶之熱穩定性與其復性難易之關係,中原大學碩士論文(2002) 曾資棟,差向異構酶包涵體復性最適化,中興大學碩士論文(2003) 林靜宜,以氫氧磷灰石固定化金屬親和吸附材進行蛋白質復性之研究(2004) 簡佩洵,以連續式饋料法進行蛋白質復性之研究(2004) Amulya K. Panda (2003) Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli, Adv. Biochem. Engin./Biotechnol. ,85:43~93 Anton P.J. Middelberg(2002) Preparative protein refolding, TRENDS in Biotechnology , 20 (10) Batas B.,Schiraldic C., Chaudhuri(1999), Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography, J. of Biotechnology,68:149~158 Boris Batas and Julian B. Chaudhuri(1996) Protein Refolding at High Concentration Using Size-Exclusion Chromatography, Biotechnology and Bioengineering ,50:16~23 Biochemistry, second edition by Garrett & Grisham Cleland,J.L.,Wang,D.I.(1990).Refolding and aggregation of bovine carbonic anhydrase B:quasi-elastic light scattering analysis. Biochemistry.29(50):11072-8 Cleland,J.L.,Builder,S.E.,Swartz,J.R.,Winkler,M.Chang,T.Y.,Wang,D.I.C.(1992) Polyethylene Glycol Enhanced Refolding of Bovine Carbonic Anhydrase B,The Journal of biological chemistry, 267(19): 13327~13334 Eliana De Bernardez Clark(2001) Protein refolding for industrial processes, Current Opinion in Biotechnology,12:202~207 Fischer B., Summer I.(1993) Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in E.coli as inclusion bodies. Biotech. Bioeng,41(1) Georgiou ,G. ,Valax ,P. (1999) Isolation inclusion bodies from bacteria, Methods Enzymol , 309:48~58 Hiroyasu Inoue, Saori Takahashi, Kiyoshi Fukui, and Yoshihiro Miyake(1991) Leucine Zipper Motif in Porcine Renin-binding Protein (RnBP) and Its Relationship to the Formation of an RnBP-Renin Heterodimer and an RnBP Homodime,J. Biol. Chem. 266:11896~11900. Jhansi Maachupalli-Reddy, Brian D. Kelley, Eliana De Bernardez Clark(1997) Effect of Inclusion Body Contaminants on the Oxidative Renaturation of Hen Egg White Lysozyme, Biotechnol. Prog.,13: 144~150 Katoh,S.,Terashima M.,Kishida H.,Yagi H.(1997) Refolding efficiency of lysozyme in fed-batch system,J.Chem. Eng. Jpn., 30:964~966 Kurucz I, Titus JA, Jost CR, Segal DM.(1995) Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies, Mol Immunol. ,32(17-18):1443~52. Kouhei Tsumoto, Mitsuo Umetsu, Izumi Kumagai,Daisuke Ejima,John S. Philo,Tsutomu Arakawa(2004) Role of Arginine in Protein Refolding, Solubilization, and Purification, Biotechnol. Prog., 20:1301-1308 Leandro B. Rodr guez-Aparicio, Miguel A. Ferrero, Beatriz Revilla-Nuin,Honorina Martnez-Blanco, Angel Reglero(1999) Determination of dierent amino sugar 2-epimerase activities by coupling to N-acetylneuraminate synthesis Biochimica et Biophysica Acta 1428 :305~313 Maeda,Y.,Koga,H.Ueda,T.,Imoto ,T.(1995) Effective renaturation of reduced l ysozyme by gentle removal of urea, Protein Eng.,8:201~205 Mamoru, M.,Daisuke,N.,Tomoya,S.(1992) Different between guandinium chloride and urea as denaturants of globular protein:The possibility of application to improve refolding process,Chem. Pharm. Bull.,40:550~552 Maru I., Ohta Y., Murata K. & Tsukada Y. , (1996a) Molecular cloning and identification of of N-acyl-D-glucosamine 2-epimerase from porcine kidney as a renin-binding protein. J. Biol. Chem. 271:16294~16299. Mckee T.,Mckee J.R.(1996) Biochemistry2nd ed., McGraw-Hill Publishers:New York Meng F, Park Y, Zhou H.(2001) Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase, Int J Biochem Cell Biol. ,3(7):701~709 Michael Cardamone, Nirdosh K. Puri, Mal R. Brandon(1995) Comparing the Refolding and Reoxidation of Recombinant Porcine Growth Hormone from a Urea Denatured State and from Escherichia coli Inclusion Bodies ,Biochemistry ,34(17):5773-5794 Mitraki A., J. King(1989) Protein folding intermediates and inclusion body formation, Bio/technology, 7:690-697 Misawa,S.,Kumagai,I.(1999) Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies, Biopolymers (Peptide Science) 51:297~307 Mukhopadhyay A(1997) Inclusion bodies and purification of proteins in biologically active forms. Adv. Biochem. Eng. Biotechnol., 56:61~109 Morganti, S.,Ueda,E.K.M,Gout,P.W. (2003) Current and propective application of metal ion-protein binding.Journal of Chromatography A.988:1-23. Neurath,H.,Greenstein,J.P.,Putnam,F. W.,Erickson J.O.(1943) The Chemistry of protein denaturation ,Chem. Rev.,32:157~265 Rainer Rudolph,Hauke Lilie(1996) In vitro folding of inclusion body proteins, FASEB J.,10(49) Rebecca McNamara , 5/24/05 http://som.flinders.edu.au/FUSA/HUMANPHYS/Parkinsons/Proteomics.html Sadana, A.(1995) Review, protein refolding and inactivation during bioseparation:Bioseparation implication , Biotechnol. Bioeng.,48,481 Saori Takahashi,Hiroyasu Inoue, Kiyoshi Fukui, and Yoshihiro Miyake(1991) Leucine Zipper Motif in Porcine Renin-binding Protein (RnBP) and Its Relationship to the Formation of an RnBP-Renin Heterodimer and an RnBP Homodime,J. Biol. Chem. 266:11896~11900. Takafumi Itoh , Bunzo Mikami, Isafumi Maru, Yasuhiro Ohta, Wataru Hashimoto and Kousaku Murata(2000) Crystal Structure of N-Acyl-D-glucosamine 2-Epimerase from Porcine Kidney at 2.0 A Resolution,J. Mol. Biol.,303:733~744 Tsumoto K, Ejima D, Kumagai I, Arakawa T.(2003) Practical considerations in refolding proteins from inclusion bodies, Protein Expr Purif. ,28(1):1~8 West,S.M.,Chaudhuri,J. B.,and Howell J.A(1998) Improved Protein Refolding Using Hollow-Fibre Membrane Dialysis,Biotechnology and bioengineering,57(5):590~599 Wilkinson DL, Harrison RG(1991)Predicting the solubility of recombinant proteins in Escherichia coli,Biotechnology(NY). 9(5):443~448 Xie Y.,Wetlaufer D B.(1996) Control of aggregation in protein refolding: the temperature-leap tactic, Protein Science,5(3)
摘要: 利用大腸桿菌生產重組差向異構酶,在大量表現時會在胞內形成大量不溶且不具活性的內函體(inclusion body),所以為使內函體回復成具天然活性的蛋白質,故發展出一個能在復性過程中減少蛋白質集結現象發生之最適化內函體復性程序。本研究以Ni-NTA固定化金屬親和吸附材進行復性,並探討其與直接稀釋法對差向異構酶之復性效果。研究發現,以稀釋法復性所得之差向異構酶具有活性,其與天然差向異構酶相對比活性為11.92%,可溶蛋白回率為42%。而利用固定化金屬方法復性雖有較佳的可溶蛋白回收率為73%,但其可溶蛋白卻都無活性。於是更進一步地針對復性環境的pH值、鹽類濃度來改善可溶蛋白回收率及活性。實驗結果,改變復性pH環境於pH=7時有75.9%可溶蛋白回收率,但還是無法使其有活性。至於添加NaCl於復性緩衝液,其濃度愈高時,可溶蛋白回收率愈低,但對活性而言亦無幫助。
Over-expression of recombinant GlcNAc 2-epimerase in Escherichia coli., led to the accumulation of insoluble and inactive proteins inclusion bodies. In this study, the application of Ni-NTA as immobilized metal affinity adsorbents was used for on column refolding. The yield of soluble protein of the on-column refolding process, 73%, is significantly higher than that of the dilution method, 42%. However, the refolded protein did not exhibit epimerase activity. The results of dynamic light scattering and size-exclusion chromatography indicated that the refolded protein aggregated into inactive oligomers. While the extent of oligomer formation was found to be essentially independent of pH, the increase in salt concentration led to a marked increase in aggregation. These results indicate the formation of epimerase oligomer is mediated by hydrophobic interaction.
URI: http://hdl.handle.net/11455/3643
其他識別: U0005-2408200718351700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2408200718351700
Appears in Collections:化學工程學系所

文件中的檔案:

取得全文請前往華藝線上圖書館

Show full item record
 
TAIR Related Article
 
Citations:


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