Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2015, Vol. 35 Issue (3): 84-91    DOI: 10.13523/j.cb.20150312
    
Liquid Chromatographic Refolding of Proteins and Process Evaluation
GAO Wen1, GAO Xiang-dong1, LU Xiao-dong2, XU Chen2
1. College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China;
2. Beijing Tri-Prime Genetic Engineering Incorporated, Beijing 102600, China
Download: HTML   PDF(616KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Refolding process of proteins is a hot spot in the area of recombinant protein drug. Dilution or dialysis requires a large amount of refolding buffer and results in a low yield. Liquid chromatographic refolding has successfully proven its capability to achieve a high yield at high protein concentrations and it is easy for productive scaling up. Partial purification of recombinant protein could also be achieved simultaneously. The recent development of liquid chromatographic refolding process is reviewed in this paper. The principles, advantages and disadvantages are discussed with respect to various chromatographic refolding processes, including size exclusion chromatography, ion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The refolding process evaluation procedures are summarized, including characterization of refolding proteins, recovery yield and industrial viability.



Key wordsProteins      Liquid chromatography      Refolding      Process evaluation     
Received: 09 December 2014      Published: 25 March 2015
ZTFLH:  Q812  
Cite this article:

GAO Wen, GAO Xiang-dong, LU Xiao-dong, XU Chen. Liquid Chromatographic Refolding of Proteins and Process Evaluation. China Biotechnology, 2015, 35(3): 84-91.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20150312     OR     https://manu60.magtech.com.cn/biotech/Y2015/V35/I3/84


[1] Oliveberg M,Wolynes P G.The experimental survey of protein-folding energy landscape.Quarterly Reviews of Biophysics,2005,38(3):245-288.

[2] Krishna M M, Lin Y, Mayne L,et al. Intimate view of a kinetic protein folding intermediate: residue-resolved structure, interactions, stability, folding and unfolding rates, homogeneity.Journal of Molecular Biology,2003,334(3):501-513.

[3] Clark E D.Refolding of recombinant proteins.Current Opinion in Biotechnology,1998,9(2):157-163.

[4] Hudson D A, Gannon S A,Thorpe C.Oxidative protein folding:From thiol–disulfide exchange reactions to the redox poise of the endoplasmic reticulum. Free Radical Biology and Medicine,2015,3:171-182.

[5] Anfinsen C B,Redfield R R,Choate W L,et al.Studies on the gross structure,cross-linkages,and terminal sequences in ribonuclease.The Journal of Biological Chemistry,1954,207(1):201-210.

[6] Frerman F E,Duncombe G R. Studies on the subunits of Escherichia coli coenzyme A transferase:reconstitution of an active enzyme.Biochimica et Biophysica Acta,1979,580(2):289-297.

[7] Hagen A J,Hatton T A,Wang D I.Protein refolding in reversed micelles.Biotechnology and Bioengineering,1990,35(10):955-965.

[8] 耿信笃,冯文科,常建华,等.用制备型高效疏水色谱复性和预分离重组人干扰素-gamma.高技术通讯,1991,1(7):1-4. Geng X D,Feng W K,Chang J H,et al.Refolding and pre-separation of recombinant human interferon-gamma using preparative high performance liquid chromatography. High Technology Letters,1991,1(7):1-4.

[9] Werner M H,Clore G M,Gronenborn A M,et al.Refolding proteins by gel filtration chromatography.FEBS Letters,1994,345(2-3):125-130.

[10] Suttnar J,Dyr J E,Hamsikova E,et al.Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger.Jouranl of Chromatography B,1994,656(1):123-126.

[11] Taguchi H,Makino Y,Yoshida M.Monomeric chaperonin-60 and its 50-kDa fragment posses the ability to interact with non-native proteins,to suppress aggregation,and to promote protein folding.The Journal of Biological Chemistry,1994,269(11):8529-8534.

[12] Phadtare S,Fisher M T,Yarbrough L R. Refolding and release of tubulins by a functional immobilized groEL column. Biochimica et Biophysica Acta,1994,1208(1):189-192.

[13] Forciniti D.Protein refolding using aqueous two-phase systems.Journal of Chromatography A,1994,668(1):95-100.

[14] Gorovits B M,Horowitz P M.High hydrostatic pressure can reverse aggregation of protein folding intermediates and facilitate acquisition of native structure.Biochemistry,1998,37(17):6132-6135.

[15] Cho T H,Ahn S J,Lee E K.Refolding of protein inclusion bodies directly from E.coli homogenate using expanded bed adsorption chromatography.Bioseparation,2001,10(4-5):189-196.

[16] Chiku H,Matsui M,Murakami S,et al. Zeolites as new chromatographic carriers for proteins:easy recovery of proteins adsorbed on zeolites by polyethylene glycol.Analytical Biochemistry,2003,318(1):80-85.

[17] Chow M K, Amin A A, Fulton K F, et al. The REFOLD database: a tool for the optimization of protein expression and refolding.Nucleic Acids Research,2006,34(Database issue):D207-D212.

[18] Chen Z,Cui Y F,Leong Y A,et al.Efficient production of recombinant IL-21 proteins for pre-clinical studies by a two-step dilution refoldinong method.International Immunopharmacology,2013,16(3):376-381.

[19] Zhao D W,Liu Y D,Wang Y J,et al.Membrane combined with hydrophilic macromolecules enhances protein refolding at high concentration.Process Biochemisty.2014,49(7):1129-1134.

[20] Englander S W,Mayne L.The nature of protein folding pathways.Proceedings of the National Academy Sciences of the United States of America,2014,11(45):15873-15880.

[21] Yuan J,Zhou H F,Yang Y C,et al.Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.Biomedical Chromatography,2014,doi:10.1002/bmc.3358.

[22] Rane A M,Jonnalagadda S,Li Z Y. On-column refolding of bone morphogenetic protein-2 using cation exchange resin.Protein Expression and Purification,2013,90(2):135-140.

[23] Sharapova O A,Yurkova M S,Laurinavichyute D K,et al.Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography.Journal of Chromatography A,2011,1218(31):5115-5119.

[24] Simpson R J.Purifying Proteins for Proteomics:A Laboratory Manual.New York:Cold Spring Harbor Laboratory Press,2004: 305,489-516.

[25] Antonio-Perez A,Ramon-Luing L A,Ortega-Lopez J.Chromatographic refolding of rhodanese and lysozyme assisted by the GroEL apical domain,DsbA and DsbC immobilized on cellulose.Journal of Chromatography A,2012,1248:122-129.

[26] Wang C Z,Geng X D.Refolding and purification of recombinant human granulocyte colony-stimulating factor using hydrophobic interaction chromatography at a large scale.Process Biochemistry,2012,47(12):2262-2266.

[27] Englander S W,Mayne L.The nature of protein folding pathways.Proceedings of the National Academy Sciences of the United States of America,2014,11(45):15873-15880.

[28] Chen Y C,Lin W Y,Wu K W,et al.Efficient lysozyme refolding at a high final concentration and a low dilution factor.Process Biochemistry,2012,47(12):1883-1888.

[29] Wang S S,Chang C K,Peng M J,et al.Effect of glutathione redox system on lysozyme refolding in size exclusion chromatography.Food and Bioproducts Processing,2006,84(1):18-27.

[30] Li M,Zhang G F,Su Z G.Dual gradient ion-exchange chromatography improved refolding yield of lysozyme.Journal of Chromatography A,2002,959(1-2):113-120.

[31] Li J J,Liu Y D,Wang F W,et al.Hydrophobic interaction chromatography correctly refolding proteins assisted by glycerol and urea gradients.Journal of Chromatography A,2004,1061(2):193-199.

[32] Dechavanne V,Barrillat N,Borlat F,et al. A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.Protein Expression and Purification,2011,75(2):192-203.

[33] Yamaguchi H,Miyazaki M. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.Biomolecules.2014,4(1):235-251.

[34] Kuo M M,Nquyen P H,Jeon Y H,et al.MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies.Microbial Cell Factories.2014,13(1):29.

[35] Han G J,Dong X Y,Sun Y. Purification effect of artificial chaperone in the refolding of recombinant ribonuclease A from inclusion bodies.Biochemical Engineering Journal.2013,77:15-19.

[36] Kuboi R,Mawatari T,Yoshimoto M.Oxidative refolding of lysozyme assisted by negatively charged liposomes:relationship with lysozyme-mediated fusion of liposomes.Journal of Bioscience and Bioengineering.2000,90(1):14-19.

[37] Ishida H,Garcia-Herrero A,Vogel H J.The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition.Biochimiaca et Biophysica Acta.2014,1838(12):3014-3024.

[38] Xu J,Li J,Wu X,et al. Expression and refolding of bioactive α-bungarotoxin V31 in E. coli.Protein Expression and Purification.2015,doi:10.1016/j.pep.

[39] Niforou K,Cheimonidou C,Trougakos I P.Molecular chaperones and proteostasis regulation during redox imbalance.Redox Biology.2014,2:323-332.

[40] Tsumoto K,Ejima D,Kumagai I,et al.Practical considerations in refolding proteins from inclusion bodies.Protein Expression and Purification,2003,28(1):1-8.

[41] Chen Y,Leong S S.Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography.Journal of Chromatography A.2009,1216(24):4877-4886.

[42] 王静岩,朱胜庚,徐长法.生物化学.上册-3版.北京:高等教育出版社,2002:197-201. Wang J Y,Zhu S G,Xu C F.Biochemistry I.3rd ed.Beijing:Higher Education Press,2002:197-201.

[43] Denslow N D,Wingfield P T,Rose K. Overview of the characterization of recombinant proteins.Current Protocols in Protein Science,2001,doi:10.1002/0471140864.ps0701s00.

[44] Gull N,Mir M A,Khan J M,et al.Refolding of bovine serum albumin via artificial chaperone protocol using gemini surfactants.Journal of Colloid and Interface Science.2011,364(1):157-162.

[45] Chura-Chambi R M,Nakajima E,Carvalho R R,et al.Refolding of the recombinant protein Sm29,a step toward the production of the vaccine candidate against schistosomiasis.Journal of Biotechnology,2013,168(4):511-519.

[46] Linke T,Aspelund M T,Thompson C,et al. Development and scale-up of a commercial fed batch refolding process for an anti-CD22 two chain immunotoxin.Biotechnology Progress.2014,30(6):1380-1389.

[47] Ogura K,Kobashigawa Y,Saio T,et al.Practical applications of hydrostatic pressure to refold proteins from inclusion bodies for NMR structural studies.Protein Engineering,Design & Selection.2013,25(6):409-416.

[48] Barends T R,Foucar L,Botha S,et al. De novo protein crystal structure determination from X-ray free-electron laser data.Nature.2014,505(7482):244-247.

[49] Gallagher S R. One-dimensional electrophoresis using nondenaturing conditions.Current Protocols in Protein Science,2001,doi:10.1002/0471142727.mb1002bs47.

[50] Alsemgeest J,Old J M,Young L J.The macropod type 2 interferon gene shares important regulatory and functionally relevant regions with eutherian IFN-γ.Molecular Immunology,2015,63(2):297-304.

[51] Pizarro S A,Gunson J,Field M J,et al.High-yield expression of human vascular endothelial growth factor VEGF165 in Escherichia coli and purification for therapeutic applications.Protein Expression and Purification,2010,72(2):184-193.

[52] Coutard B,Danchin E G J,Oubelaid R,et al.Single pH buffer refolding screen for protein from inclusion bodies.Protein Expression and Purification.2012,82(2):352-359.

[53] Dechavanne V,Barrillat N,Borlat F,et al.A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimiza the refolding conditions.Protein Expression and Purification.2011,75(2):192-203.

[1] FENG Zhao,LI Jiang-hao,WANG Jia-hua. Functional Analysis of RpRPL22, a Ribosomal Protein Homologous Gene, in the Symbiotic Nodulation Process of Robinia Pseudoacacia[J]. China Biotechnology, 2021, 41(7): 10-21.
[2] XUE Rui,YAO Lin,WANG Rui,LUO Zheng-shan,XU Hong,LI Sha. Advances and Applications of Recombinant Mussel Foot Proteins[J]. China Biotechnology, 2020, 40(11): 82-89.
[3] DAO Feng-ting,YANG Lu,WANG Ya-zhe,CHANG Yan,YUAN Xiao-ying,LI Ling-di,CHEN Wen-min,LONG Ling-yu,LIU Yan-rong,QIN Ya-zhen. Characteristics and Prognostic Significance of Ki-67 Expression at diagnosis in Adult t(8;21) Acute Myeloid Leukemia[J]. China Biotechnology, 2019, 39(9): 11-18.
[4] Gong CHENG,Si-ming JIAO,Li-shi REN,Cui FENG,Yu-guang DU. Preparation and Composition Analysis of Chitooligosaccharides with Low Degree of Deacetylation by Hydrolysis of Bacillus subtilis Chitosanase[J]. China Biotechnology, 2018, 38(9): 19-26.
[5] Yue ZHAO,Hao WU,Jian-jun QIAO. Research on the Regulatory Mechanisms of Bacterial Cell Wall Growth[J]. China Biotechnology, 2018, 38(8): 92-99.
[6] Qing-meng LI,Sheng-tao LI,Ning WANG,Xiao-dong GAO. Expression, Purification and Activity Assay of Yeast α-1,2 Mannosyltransferase Alg11[J]. China Biotechnology, 2018, 38(6): 26-33.
[7] WANG Yan-hong,LIU Yan-shuang,SHI De-xi,ZHU Bao-guo,LV Bao-lei,FU Shi-yu,XU Miao,WANG We,YIN Kui-de. Functional Identification of Na +/H + Antiporter in Novel YdjM Superfamily Members[J]. China Biotechnology, 2018, 38(12): 32-40.
[8] XU Li, JI Cai-xia, LIU Xiao-hua, YU Ting-ting, LUO Jin-yong. Effects of DLX1 on BMP9-induced Osteogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells[J]. China Biotechnology, 2017, 37(10): 8-15.
[9] LIN You-hong, CHENG Xia-ying, YAN Yi-wen, LIANG Zong-suo, YANG Zong-qi. Expression and Optimization Strategy of Recombinant Proteins in Chlamydomonas Chloroplast[J]. China Biotechnology, 2017, 37(10): 118-125.
[10] CHEN Bing, KONG Ling-jiao, LEI Jin-xia, SHEN Lu, ZHANG Cai, WANG Jin-hua. BMP9 Induces Osteogenic/odontogenic Differentiation of ISCAP through the Smad Pathway[J]. China Biotechnology, 2016, 36(8): 16-22.
[11] XUE Ling, LIU Jiang-ning, ZHANG Yao, ZHANG Chun, WANG Qi, QIN Chuan, LIU Yong-dong, SU Zhi-guo. Affinity Purification of Enterovirus 71 Fused Multi-Epitope Protein Antigen and Assembling It as Virus-like Particles in Vitro[J]. China Biotechnology, 2016, 36(7): 34-40.
[12] LI Ya-sha, LIU Xing, BI Yang, YANG Ke, ZHAO Li, GONG Meng-jia, GUO Qi. BMP9 Induced the Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in vitro and vivo[J]. China Biotechnology, 2016, 36(5): 20-26.
[13] YU Xiu-min, YUE Wen-ran, ZHANG Yan-na, YANG Fei-yun, WANG Rui-gang, LI Guo-jing, YANG Qi. Heterologous of CkLEA1 Gene Enhanced Tolerance to Abiotic Stress in Arabidopsis[J]. China Biotechnology, 2016, 36(10): 28-34.
[14] LIU Dong-mei, SUN Jia-nan, ZOU Jia-ning, LIU Ming-wei, SUN Lu, LIU Qiong-ming. Screening Interacting Proteins for BAG Domains of BAG Family Proteins by GST pull-down Coupled with LC-MS/MS[J]. China Biotechnology, 2015, 35(4): 1-10.
[15] LI Hui, XUE Wei, SUN Xue-song. Ferrichrome Binding Characteristics of Wild Type-and Mutant FtsBs in Streptococcus pyogenes[J]. China Biotechnology, 2015, 35(10): 32-38.