折叠酶DsbA的制备及协助蛋白质体外复性的研究
摘要
如何对富含二硫键的包涵体蛋白进行高效的体外复性是重组蛋白生产所面临的巨大挑战。DsbA是大肠杆菌周质胞腔中辅助多种含有二硫键的蛋白质正确折叠并具有生物学活性的一种二硫键异构酶。本文将DsbA质粒成功转化后，对转化子进行了发酵和纯化的优化，然后将制备的重组折叠酶DsbA初步应用于模型蛋白溶菌酶的体外复性之中。
首先，将含编码dsbA基因的质粒pAVD63转化到E．coli BL21(DE3)，目标蛋白DsbA获得了可溶性表达。
然后，利用统计实验设计的方法将生产重组DsbA的发酵过程进行了优化。通过Plackett-Burman设计挑选出了对DsbA表达量影响较大的4个因素，利用杂合设计进行实验，并通过拟合得到了响应曲面函数，但该函数的驻点是鞍点，因此不具有全局的极值。通过约束优化得到了较佳的实验点，在该实验点下DsbA的表达量比基本培养条件下提高了50.14％。
再次，离心收集细胞后进行超声波破胞，将细胞破碎的清液经过离子交换层析得到纯化的DsbA。为提高DsbA分离纯化的效率、回收率并缩短DsbA的制备周期，对离子交换层析过程利用Box-Behnken设计进行了统计优化，考察了pH值、洗脱梯度、初始盐浓度和上样量的影响。以DsbA洗脱峰面积为响应值，拟合出响应面函数，并利用多目标规划中的功效系数法综合考虑以上3个目标，得到了较优的实验条件为：20mL Q Sepharose Fast Flow凝胶柱，pH值为8.3，洗脱高盐含量(B液所占比例)为5.88％，初始盐浓度为16.7mM，上样量为9.25mL。验证实验表明，得到的DsbA洗脱峰面积为4301.8mAU·mL，与模型预测的的相对误差仅为±2.4％。最终纯化后可得到377.9±1.74mg DsbA／L发酵液，蛋白收率为96.8％，纯度大于95％。通过肽质量指纹谱分析所得目标产物为高纯度的DsbA，分子量为23kD。
最后，将DsbA应用于模型蛋白溶菌酶的复性过程中。利用Plakett-Burman设计考察了影响复性后溶菌酶蛋白回收和比活的9个因素，筛选出5个重要因素：初始溶菌酶浓度、尿素浓度、KCl浓度、GSSG浓度、GSH与GSSG摩尔比，并进一步对这5个重要因素进行了考察。在优化的复性条件下，当初始溶菌酶浓度为250μg／mL时，DsbA介导体系复性效果大大高于直接稀释复性的结果，溶菌酶活性回收率从44.52％提高到75.85％，为富含二硫键的包涵体蛋白体外复性提供了一种新的方法。
How to refold inclusion body proteins with many pairs of disulfide bonds into natural conformation with high efficiency is a challengeable problem at present. DsbA, existing in the periplasm of E. coli, is a kind of disulfide bond isomerase, which assists the folding of the disulfide bond containing proteins to achieve biological activities. In the thesis, the plasmid containing the gene of dsbA was successfully transformed into E.coli, the culture conditions of the DsbA transformant in the fermentation and purification were optimized, then the purified DsbA was applied to assist the renaturation of lysozyme in vitro.
Firstly, the plasmid pAVD63 coded with dsbA gene was tramsformed into E.coli BL21 (DE3) and the target protein was expressed intracellularly.
Secondly, statistical experimental designs were used to optimize the fermentation process of recombinant DsbA in E.coli. Four most important parameters, which affect the DsbA expression, were screened among 17 parameters by Plackett-Burman design. Then hybrid design was applied to fit a response surface function, where stationary point was a saddle point with neither global maximum nor minimum. Finally, the optimal settings of fermentation parameters were calculated using the nonlinear constrained optimization method. Verification experiment showed that the settings resulted in 50.14% higher expression after the optimization.
Thirdly, after the cells were collected by centrifugation and disrupted by ultrasonic with ice bath, DsbA was purified by ion exchange chromatograpy (IEC). In order to improve the efficiency and DsbA recovery of the purification process and shorten the production period, the IEC process was optimized by Box-Behnken design, the influence of pH, step of elution, initial salt concentration and loading amout were studied. The area of DsbA elution peak was set as the response to fit the response function, and three objects were integrated together by effectiveness coefficient method in MOP (Mutiple Objective Programming) and finally the optimized process was achieved: 20mL Q Sepharose Fast Flow gel packed column, pH 8.3, elution at step of 5.88% (percent of the buffer B, pH 8.3, 16.7mM Tris-HCl, 2M KCl), initial salt concentration 16.7mM, loading amount 9.25mL. The verification experiment showed the setting resulted in 4301.8 mAU·mL of the area of the DsbA elution peak, while the error was only ±2.4% compared with the predicted value. In the whole process, 377.9±1.74mg purified DsbA per liter fermentation broth was obtained, and
the protein recovery was 96.8%, the purity was more than 95%. The high purity of DsbA was identified by peptide mass finger print (PMF), and the molecular weight was ascertained to be 23kD.
Finally, DsbA was applied to assist the refolding of the model protein lysozyme. Plackett-Burman design was employed to investigate 9 factors which influenced the lysozyme protein recovery and specific activity, and 5 most important factors were screened out: initial lysozyme concentration, urea concentration, KC1 concentration, GSSG concentration and the molar ratio of GSH to GSSG. Furthermore, these five significant factors were studied in details. At optimized experimental conditions, the activity recovery was increased from 44.52% to 75.85% in this DsbA mediated refolding system compared with direct dilution refolding when the initial lysozyme concentration was 250 μg/mL, This research provides an alternative method for the refolding of recombinant proteins expressed as inclusion bodies in vitro with many pairs of disulfide bonds.
首先，将含编码dsbA基因的质粒pAVD63转化到E．coli BL21(DE3)，目标蛋白DsbA获得了可溶性表达。
然后，利用统计实验设计的方法将生产重组DsbA的发酵过程进行了优化。通过Plackett-Burman设计挑选出了对DsbA表达量影响较大的4个因素，利用杂合设计进行实验，并通过拟合得到了响应曲面函数，但该函数的驻点是鞍点，因此不具有全局的极值。通过约束优化得到了较佳的实验点，在该实验点下DsbA的表达量比基本培养条件下提高了50.14％。
再次，离心收集细胞后进行超声波破胞，将细胞破碎的清液经过离子交换层析得到纯化的DsbA。为提高DsbA分离纯化的效率、回收率并缩短DsbA的制备周期，对离子交换层析过程利用Box-Behnken设计进行了统计优化，考察了pH值、洗脱梯度、初始盐浓度和上样量的影响。以DsbA洗脱峰面积为响应值，拟合出响应面函数，并利用多目标规划中的功效系数法综合考虑以上3个目标，得到了较优的实验条件为：20mL Q Sepharose Fast Flow凝胶柱，pH值为8.3，洗脱高盐含量(B液所占比例)为5.88％，初始盐浓度为16.7mM，上样量为9.25mL。验证实验表明，得到的DsbA洗脱峰面积为4301.8mAU·mL，与模型预测的的相对误差仅为±2.4％。最终纯化后可得到377.9±1.74mg DsbA／L发酵液，蛋白收率为96.8％，纯度大于95％。通过肽质量指纹谱分析所得目标产物为高纯度的DsbA，分子量为23kD。
最后，将DsbA应用于模型蛋白溶菌酶的复性过程中。利用Plakett-Burman设计考察了影响复性后溶菌酶蛋白回收和比活的9个因素，筛选出5个重要因素：初始溶菌酶浓度、尿素浓度、KCl浓度、GSSG浓度、GSH与GSSG摩尔比，并进一步对这5个重要因素进行了考察。在优化的复性条件下，当初始溶菌酶浓度为250μg／mL时，DsbA介导体系复性效果大大高于直接稀释复性的结果，溶菌酶活性回收率从44.52％提高到75.85％，为富含二硫键的包涵体蛋白体外复性提供了一种新的方法。
How to refold inclusion body proteins with many pairs of disulfide bonds into natural conformation with high efficiency is a challengeable problem at present. DsbA, existing in the periplasm of E. coli, is a kind of disulfide bond isomerase, which assists the folding of the disulfide bond containing proteins to achieve biological activities. In the thesis, the plasmid containing the gene of dsbA was successfully transformed into E.coli, the culture conditions of the DsbA transformant in the fermentation and purification were optimized, then the purified DsbA was applied to assist the renaturation of lysozyme in vitro.
Firstly, the plasmid pAVD63 coded with dsbA gene was tramsformed into E.coli BL21 (DE3) and the target protein was expressed intracellularly.
Secondly, statistical experimental designs were used to optimize the fermentation process of recombinant DsbA in E.coli. Four most important parameters, which affect the DsbA expression, were screened among 17 parameters by Plackett-Burman design. Then hybrid design was applied to fit a response surface function, where stationary point was a saddle point with neither global maximum nor minimum. Finally, the optimal settings of fermentation parameters were calculated using the nonlinear constrained optimization method. Verification experiment showed that the settings resulted in 50.14% higher expression after the optimization.
Thirdly, after the cells were collected by centrifugation and disrupted by ultrasonic with ice bath, DsbA was purified by ion exchange chromatograpy (IEC). In order to improve the efficiency and DsbA recovery of the purification process and shorten the production period, the IEC process was optimized by Box-Behnken design, the influence of pH, step of elution, initial salt concentration and loading amout were studied. The area of DsbA elution peak was set as the response to fit the response function, and three objects were integrated together by effectiveness coefficient method in MOP (Mutiple Objective Programming) and finally the optimized process was achieved: 20mL Q Sepharose Fast Flow gel packed column, pH 8.3, elution at step of 5.88% (percent of the buffer B, pH 8.3, 16.7mM Tris-HCl, 2M KCl), initial salt concentration 16.7mM, loading amount 9.25mL. The verification experiment showed the setting resulted in 4301.8 mAU·mL of the area of the DsbA elution peak, while the error was only ±2.4% compared with the predicted value. In the whole process, 377.9±1.74mg purified DsbA per liter fermentation broth was obtained, and
the protein recovery was 96.8%, the purity was more than 95%. The high purity of DsbA was identified by peptide mass finger print (PMF), and the molecular weight was ascertained to be 23kD.
Finally, DsbA was applied to assist the refolding of the model protein lysozyme. Plackett-Burman design was employed to investigate 9 factors which influenced the lysozyme protein recovery and specific activity, and 5 most important factors were screened out: initial lysozyme concentration, urea concentration, KC1 concentration, GSSG concentration and the molar ratio of GSH to GSSG. Furthermore, these five significant factors were studied in details. At optimized experimental conditions, the activity recovery was increased from 44.52% to 75.85% in this DsbA mediated refolding system compared with direct dilution refolding when the initial lysozyme concentration was 250 μg/mL, This research provides an alternative method for the refolding of recombinant proteins expressed as inclusion bodies in vitro with many pairs of disulfide bonds.
引文
[1] Kane J. F., Hartley D. L.. Purification and analysis of recombinant protein. New York, Marcel Dekker Inc. 1991: 121-146.
[2] 凌明圣，许祥裕，丁树标．以包涵体形式存在的重组蛋白的纯化和体外折叠．中国生化药物杂志，1995，16(3)：135-139．
[3] Lilie H., Schwarz E., Rudolph R.. Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol. 1998, 9: 497-501.
[4] Maachupalli-Reddy J., Kelley B. D., Bernardez D., Clark E.. Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. Biotechnol Prog, 1997, 13(2): 144-150.
[5] Fischer B., Sumner I., Goodenough P.. Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in E. coli as inclusion bodies. Biotech Bioeng, 1993, 41(1): 3-13.
[6] 邹承鲁．第二遗传密码-新生肽及蛋白质折叠的研究．湖南科学技术出版社，1997： 60-61．
[7] Maeda Y., Ueda T., Imoto T.. Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone. Protein Eng.1996, 9: 95-100.
[8] Bernardez D., Clark E.. Refolding of recombinant proteins. Curr Opin Biotechnol,1995, 9:157-163.
[9] Batas B., Chaudhuri J. B .. Protein refolding at high concentration using size-exclusion chromatography, Biotech. Bioeng., 1996, 50: 16-23.
[10] Geng X. D., Chang X. Q.. High-performance hydrophobic interaction chromatography as a tool for protein refolding. J. Chromatogr, 1992, 599:185-194.
[11] Li M. , Zhang G. F., Su Z. G.. Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. J. Chromatogr A, 2002, 959:113-120.
[12] Zahn R.. Human Prion proteins expressed in Escherichia coli and purified by high affinity column refolding. FEBS Letters, 1997, 417, 3: 400-404.
[13] Terashima M., Suzuki K.., Katoh S.. Effective refolding of fully reduced lysozyme with a flow-type reactor. Process Biochemistry, 1996, 31(4): 341-345.
[14] Futami J., Tsushima Y., Tada H.. Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies. Journal of Biochemistry, 2000, 127: 435-441.
[15] Katoh S. , Katoh Y.. Continuous refolding of lysozyme with fed-batch addition of denatured protein solution. Process Biochemistry, 2000, 35:1119-1124.
[16] West S. M., Chaudhuri J. B., Howell J. A.. Improved protein refolding using hollow-fibre membrane dialysis. Biotechnology and Bioengineering, 1998, 57(5): 590-599.
[17] Batas B., Chaudhuri J. B.. Considerations of sample application and elution during size-exclusion chromatography-based protein refolding. Journal of Chromatography A, 1999 (864): 229-236.
[18] Werner M. H.. Refolding proteins by gel filtration chromatography. FEBS Letter, 1994, 345(2): 125-130.
[19] Muller C., Rinas U.. Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography. Journal of Chromatography A, 1999, 855: 203-213.
[20] Fahey E. M., Chaudhuri J. B.. Refolding of low molecular weight urokinase plasminogen activator by dilution and size exclusion chromatography-a comparative study. Separation Science and Technology, 2000, 35(11): 1743-1760.
[21] Liu T., Geng X. D.. The separation efficiency of biopolymers with short column in liquid chromatography. Chinese Chemical Letter, 1999, 10: 219-212.
[22] Geng X. D., Chang X. Q.. High-performance hydrophobic interaction chromatography as a tool for protein refolding. J. Chromatography, 1992, 599: 185-194.
[23] Hamaker K. H.. Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor. Biotechnology Progress, 1996, 12(2): 184-189.
[24] Suttnar J.. Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger. Journal of Chromatography B, 1994, 656(1): 123-126.
[25] Li M., Zhang G. F., Su Z. G.. Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. Journal of Chromatography A, 2002, 959: 113-120.
[26] Futami J., Tsushima Y., Tada H. Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies. Journal of Biochemistry, 2000, 127: 435-441.
[27] 张友鸿，杨克恭．蛋白质在大肠杆菌间周质中的折叠．生命的化学，2001：6-21．
[28] 王志珍．蛋白质二硫键异构酶既是酶又是分子伴侣．科学通报，1998：13-43．
[29] Dong X. Y., Yang H., Sun Y.. Lysozyme Refolding with immobilized GroEL column. Journal of Chromatography A, 2000, 878:197-204.
[30] 高永贵，溶菌酶和重组人干扰素-γ 包涵体体外折叠复性的研究．博士学位论文，浙 江大学，中国杭州，2002．
[31] Gao, Y. G., Guan, Y. X., Yao, S. J., Cho, M. G.. On-column refolding of recombinant human interferon-γ with an immobilized chaperone fragment, Biotechnology Progress, 2003, 19: 915-920.
[32] Gao Y. G., Guan Y. X., Yao S. J., Cho M. G.. Lysozyme refolding at high concentration by dilution and size-exclusion chromatography. Journal of Zhejiang University Science, 2003, 4(2): 136-141.
[33] Ahn J. H., Lee Y. P., Rhee J. S.. Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology. Journal of Biotehnology, 1997, 54: 151-160.
[34] Cardamone M., Puri N. K., Brandon M. R.. Comparing the refolding and reoxidation of recombinant porcine growth hormone from a urea denatured state and from Escherichia coli inclusion bodies. Biochemistry, 1995, 34: 5773-5794.
[35] Bam N. B., Cleland J. L., Randolph T. W.. Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants. Biotechnology Progress, 1996, 12: 801-809.
[36] Wetlaufer D. B., Xie Y.. Control of aggregation in protein refolding, a variety of surfactants promote renaturation of carbonic anhydrase Ⅱ. Protein Science, 1995, 4: 1535-1543
[37] Dorin G., McAlary P., Wong K.. Bacterial production of hydrophobic polypeptides. World (WO) Patent, 1996, 96: 39523.
[38] Kurucz I., Titus J. A., Jost C. A.. Correct disulphide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies. Molecular Immunology, 1995, 12: 1443-1452.
[39] Cerletti N., McMaster G. K., Cox D., Schmitz A., Meyback B.. Process for refolding recombinantly produced TGF-β-like proteins. US Patent 1997, 5: 650-694.
[40] Builder S., Hart R., Lester P.. Refolding of misfolded insulin-like growth factor-Ⅰ. US Patent. 1997, 5: 663-704.
[41] Kohno T., Carmichael D. F., Sommer A.. Refolding of recombinant proteins. Methods in Enzymology, 1990, 185: 187-195.
[42] 史晋辉，董晓燕，孙彦．盐酸胍浓度对变性溶菌酶复性的影响．生物化学与生物物理学报，2001，33(4)：447-451．
[43] Hevehan D., Clark E. B.. Oxidative renaturation of lysozyme at high concentrations. Biotechnology Bioengineering, 1997, 54: 221-230.
[44] 刘晓光，董晓燕．分子伴侣与蛋白质复性的简介，化学工业与工程，2000，17(2)： 120-124．
[45] Vuillard L., Madern D., Franzetti B., Rabilloud T.. Halophilic protein stabilization by mild solubilizing agents nondertergent sulfobetaines. Analytical Biochemistry, 1995, 230: 290-294.
[46] 凌明圣，许祥裕，施凤霞．聚乙二醇增加重组人粒—单系集落刺激因子复性效率的研究．药物生物技术，1997，4(1)：5-8．
[47] Shimizu H. , Fujimoto K. , Kawaguchi H.. Refolding of protein using thiol-carrying latex particle. Colloids and Surfaces A: Physiochemical and Engineering Aspects, 1999, 153: 421-427.
[48] Shimizu H., Fujimoto K., Kawaguchi H.. Improved refolding of denarured/reduced lysozyme using disulfide-carrying polymeric microspheres. Colloids and Surfaces B: Biointerfaces, 2000, 18: 137-144.
[49] Kuboi R., Morita S., Ota H., Umakoshi H.. Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems. Journal of Chromatography B, 2000, 743: 215-223.
[50] Lin S. C., Lin K. L., Chiu H. C.. Enhanced protein renaturation by temperature-responsive polymers. Biotechnology and Bioengineering, 2000, 67(5): 505-511.
[51] Debarbieux L., Beckwith J.. Electron avenue: pathways of disulfide bond formation and isomerization. Cell, 1999, 99: 117-119.
[52] Bardwell J. C. A., McGovern K., Beckwith J.. Identification of a protein required for disulfide bond formation in vivo. Cell, 1991, 67:581-589.
[53] Kamitani S., Akiyama Y., Ito K.. Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase a periplasmic enzyme. The EMBO Journal, 1992, 11(1): 57-62.
[54] Plunkett G., Burland V., Daniels D. L., Blattner F. R.. Analysis of the Escherichia coli genome. Ⅲ. DNA sequence of the region from 87.2 to 89.2 minutes, Nucleic Acids Res, 1993, 21(15): 3391-3398.
[55] Guddat L. W., Bardwell J. C., Zander T., Martin J. L.. The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding. Protein Sci, 1997, 6:1148-1156.
[56] Martin, J. L., Bardwell, J. C. A., Kurlyan, J.. Crystal structure of the DsbA protein required for disulfide bond formation in vivo, Nature, 1993, 365: 464-468.
[57] Schirra, H. J., Renner, C., Czisch, M., Huber-Wunderlich, M., Holak, T. A., Glockshuber, R... Structure of reduced DsbA from Escherichia coli in solution, Biochemistry, 1998, 37: 6263-6276.
[58] Zapun A., Creighton T. E.. Effect of DsbA on the disulfide folding of bovine pancreatic trypsin inhibitor and α-lactalbumin. Biochemistry, 1994, 33:5202-5211
[59] Maskos K., Huber-Wunderlich M., Glockshuber R.. DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo. Journal of Molecular Biology, 2003, 325:495-513
[60] Bardwell J. C. A., McGovern K., Beckwith J.. Identification of a protein required for disulfide bond formation in vivo. Cell, 1991, 67: 581-589.
[61] Zheng W. D., Quan. H., Song J. L., Yang S. L., Wang C. C.. Does DsbA have chaperone-like activity. Archives of biochemistry and biophysics, 1997, 337(2): 326-331.
[62] 李伯良，江智红．cIts857基因的克隆：修饰及温敏诱导表达载体．生物化学与生物物理学报，1994，26：389-393．
[63] Studier F. W., Moffatt B. A.. Use of Bacteriophage T7 RNA Polymerase to direct selective high level expression of cloned genes. J. Mol. Biol., 1986, 189:113-118.
[64] Mcallister W.T., Carter A. D.. Regulation of promoter selection by the bacteriophage T7 RNA polymerase in vitro. Nucl Acids.Res., 1980, 8: 4821-4825.
[65] 萨姆布鲁克J．，弗里奇E．F．，曼尼阿蒂斯T．分子克隆实验指南．科学出版社，第一版，1986：19-20．
[66] 李德葆，周雪平，许建平等．基因工程操作技术．上海科学技术出版社，1988：27-28
[67] Sambrook J., MacCallum P., Russell D.. Molecular Cloning: A laboratory manual (3rd Edition). USA: Cold Spring Harbor Laboratory Press, 2001.
[68] Haaland P. D.. Experimental design in biotechnology. New York: ASQC Quality Press, 1989: 6.
[69] Plackett R. L., Burman J. P.. The design of optimum multifactorial experiments. Biometrika, 1946, 37: 305-325.
[70] Montgomery D. C.. Design and analysis of experiments (3rd ed.). USA: John Wiley & Sons, Inc., 1991: 521-568.
[71] Roquemore K. G.. Hybrid designs for quadratic response surfaces. Technometrics, 1976, 18(4): 419-423.
[72] Altamirano M. M., Garcia C., Possani L. D., Fersht A. R.. Oxidative refolding chromatography: folding of the scorpion toxin Cn5. Nature Biotechnology, 1999, 17: 187-191.
[2] 凌明圣，许祥裕，丁树标．以包涵体形式存在的重组蛋白的纯化和体外折叠．中国生化药物杂志，1995，16(3)：135-139．
[3] Lilie H., Schwarz E., Rudolph R.. Advances in refolding of proteins produced in E. coli. Curr Opin Biotechnol. 1998, 9: 497-501.
[4] Maachupalli-Reddy J., Kelley B. D., Bernardez D., Clark E.. Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. Biotechnol Prog, 1997, 13(2): 144-150.
[5] Fischer B., Sumner I., Goodenough P.. Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in E. coli as inclusion bodies. Biotech Bioeng, 1993, 41(1): 3-13.
[6] 邹承鲁．第二遗传密码-新生肽及蛋白质折叠的研究．湖南科学技术出版社，1997： 60-61．
[7] Maeda Y., Ueda T., Imoto T.. Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone. Protein Eng.1996, 9: 95-100.
[8] Bernardez D., Clark E.. Refolding of recombinant proteins. Curr Opin Biotechnol,1995, 9:157-163.
[9] Batas B., Chaudhuri J. B .. Protein refolding at high concentration using size-exclusion chromatography, Biotech. Bioeng., 1996, 50: 16-23.
[10] Geng X. D., Chang X. Q.. High-performance hydrophobic interaction chromatography as a tool for protein refolding. J. Chromatogr, 1992, 599:185-194.
[11] Li M. , Zhang G. F., Su Z. G.. Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. J. Chromatogr A, 2002, 959:113-120.
[12] Zahn R.. Human Prion proteins expressed in Escherichia coli and purified by high affinity column refolding. FEBS Letters, 1997, 417, 3: 400-404.
[13] Terashima M., Suzuki K.., Katoh S.. Effective refolding of fully reduced lysozyme with a flow-type reactor. Process Biochemistry, 1996, 31(4): 341-345.
[14] Futami J., Tsushima Y., Tada H.. Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies. Journal of Biochemistry, 2000, 127: 435-441.
[15] Katoh S. , Katoh Y.. Continuous refolding of lysozyme with fed-batch addition of denatured protein solution. Process Biochemistry, 2000, 35:1119-1124.
[16] West S. M., Chaudhuri J. B., Howell J. A.. Improved protein refolding using hollow-fibre membrane dialysis. Biotechnology and Bioengineering, 1998, 57(5): 590-599.
[17] Batas B., Chaudhuri J. B.. Considerations of sample application and elution during size-exclusion chromatography-based protein refolding. Journal of Chromatography A, 1999 (864): 229-236.
[18] Werner M. H.. Refolding proteins by gel filtration chromatography. FEBS Letter, 1994, 345(2): 125-130.
[19] Muller C., Rinas U.. Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography. Journal of Chromatography A, 1999, 855: 203-213.
[20] Fahey E. M., Chaudhuri J. B.. Refolding of low molecular weight urokinase plasminogen activator by dilution and size exclusion chromatography-a comparative study. Separation Science and Technology, 2000, 35(11): 1743-1760.
[21] Liu T., Geng X. D.. The separation efficiency of biopolymers with short column in liquid chromatography. Chinese Chemical Letter, 1999, 10: 219-212.
[22] Geng X. D., Chang X. Q.. High-performance hydrophobic interaction chromatography as a tool for protein refolding. J. Chromatography, 1992, 599: 185-194.
[23] Hamaker K. H.. Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor. Biotechnology Progress, 1996, 12(2): 184-189.
[24] Suttnar J.. Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger. Journal of Chromatography B, 1994, 656(1): 123-126.
[25] Li M., Zhang G. F., Su Z. G.. Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. Journal of Chromatography A, 2002, 959: 113-120.
[26] Futami J., Tsushima Y., Tada H. Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies. Journal of Biochemistry, 2000, 127: 435-441.
[27] 张友鸿，杨克恭．蛋白质在大肠杆菌间周质中的折叠．生命的化学，2001：6-21．
[28] 王志珍．蛋白质二硫键异构酶既是酶又是分子伴侣．科学通报，1998：13-43．
[29] Dong X. Y., Yang H., Sun Y.. Lysozyme Refolding with immobilized GroEL column. Journal of Chromatography A, 2000, 878:197-204.
[30] 高永贵，溶菌酶和重组人干扰素-γ 包涵体体外折叠复性的研究．博士学位论文，浙 江大学，中国杭州，2002．
[31] Gao, Y. G., Guan, Y. X., Yao, S. J., Cho, M. G.. On-column refolding of recombinant human interferon-γ with an immobilized chaperone fragment, Biotechnology Progress, 2003, 19: 915-920.
[32] Gao Y. G., Guan Y. X., Yao S. J., Cho M. G.. Lysozyme refolding at high concentration by dilution and size-exclusion chromatography. Journal of Zhejiang University Science, 2003, 4(2): 136-141.
[33] Ahn J. H., Lee Y. P., Rhee J. S.. Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology. Journal of Biotehnology, 1997, 54: 151-160.
[34] Cardamone M., Puri N. K., Brandon M. R.. Comparing the refolding and reoxidation of recombinant porcine growth hormone from a urea denatured state and from Escherichia coli inclusion bodies. Biochemistry, 1995, 34: 5773-5794.
[35] Bam N. B., Cleland J. L., Randolph T. W.. Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants. Biotechnology Progress, 1996, 12: 801-809.
[36] Wetlaufer D. B., Xie Y.. Control of aggregation in protein refolding, a variety of surfactants promote renaturation of carbonic anhydrase Ⅱ. Protein Science, 1995, 4: 1535-1543
[37] Dorin G., McAlary P., Wong K.. Bacterial production of hydrophobic polypeptides. World (WO) Patent, 1996, 96: 39523.
[38] Kurucz I., Titus J. A., Jost C. A.. Correct disulphide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies. Molecular Immunology, 1995, 12: 1443-1452.
[39] Cerletti N., McMaster G. K., Cox D., Schmitz A., Meyback B.. Process for refolding recombinantly produced TGF-β-like proteins. US Patent 1997, 5: 650-694.
[40] Builder S., Hart R., Lester P.. Refolding of misfolded insulin-like growth factor-Ⅰ. US Patent. 1997, 5: 663-704.
[41] Kohno T., Carmichael D. F., Sommer A.. Refolding of recombinant proteins. Methods in Enzymology, 1990, 185: 187-195.
[42] 史晋辉，董晓燕，孙彦．盐酸胍浓度对变性溶菌酶复性的影响．生物化学与生物物理学报，2001，33(4)：447-451．
[43] Hevehan D., Clark E. B.. Oxidative renaturation of lysozyme at high concentrations. Biotechnology Bioengineering, 1997, 54: 221-230.
[44] 刘晓光，董晓燕．分子伴侣与蛋白质复性的简介，化学工业与工程，2000，17(2)： 120-124．
[45] Vuillard L., Madern D., Franzetti B., Rabilloud T.. Halophilic protein stabilization by mild solubilizing agents nondertergent sulfobetaines. Analytical Biochemistry, 1995, 230: 290-294.
[46] 凌明圣，许祥裕，施凤霞．聚乙二醇增加重组人粒—单系集落刺激因子复性效率的研究．药物生物技术，1997，4(1)：5-8．
[47] Shimizu H. , Fujimoto K. , Kawaguchi H.. Refolding of protein using thiol-carrying latex particle. Colloids and Surfaces A: Physiochemical and Engineering Aspects, 1999, 153: 421-427.
[48] Shimizu H., Fujimoto K., Kawaguchi H.. Improved refolding of denarured/reduced lysozyme using disulfide-carrying polymeric microspheres. Colloids and Surfaces B: Biointerfaces, 2000, 18: 137-144.
[49] Kuboi R., Morita S., Ota H., Umakoshi H.. Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems. Journal of Chromatography B, 2000, 743: 215-223.
[50] Lin S. C., Lin K. L., Chiu H. C.. Enhanced protein renaturation by temperature-responsive polymers. Biotechnology and Bioengineering, 2000, 67(5): 505-511.
[51] Debarbieux L., Beckwith J.. Electron avenue: pathways of disulfide bond formation and isomerization. Cell, 1999, 99: 117-119.
[52] Bardwell J. C. A., McGovern K., Beckwith J.. Identification of a protein required for disulfide bond formation in vivo. Cell, 1991, 67:581-589.
[53] Kamitani S., Akiyama Y., Ito K.. Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase a periplasmic enzyme. The EMBO Journal, 1992, 11(1): 57-62.
[54] Plunkett G., Burland V., Daniels D. L., Blattner F. R.. Analysis of the Escherichia coli genome. Ⅲ. DNA sequence of the region from 87.2 to 89.2 minutes, Nucleic Acids Res, 1993, 21(15): 3391-3398.
[55] Guddat L. W., Bardwell J. C., Zander T., Martin J. L.. The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding. Protein Sci, 1997, 6:1148-1156.
[56] Martin, J. L., Bardwell, J. C. A., Kurlyan, J.. Crystal structure of the DsbA protein required for disulfide bond formation in vivo, Nature, 1993, 365: 464-468.
[57] Schirra, H. J., Renner, C., Czisch, M., Huber-Wunderlich, M., Holak, T. A., Glockshuber, R... Structure of reduced DsbA from Escherichia coli in solution, Biochemistry, 1998, 37: 6263-6276.
[58] Zapun A., Creighton T. E.. Effect of DsbA on the disulfide folding of bovine pancreatic trypsin inhibitor and α-lactalbumin. Biochemistry, 1994, 33:5202-5211
[59] Maskos K., Huber-Wunderlich M., Glockshuber R.. DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo. Journal of Molecular Biology, 2003, 325:495-513
[60] Bardwell J. C. A., McGovern K., Beckwith J.. Identification of a protein required for disulfide bond formation in vivo. Cell, 1991, 67: 581-589.
[61] Zheng W. D., Quan. H., Song J. L., Yang S. L., Wang C. C.. Does DsbA have chaperone-like activity. Archives of biochemistry and biophysics, 1997, 337(2): 326-331.
[62] 李伯良，江智红．cIts857基因的克隆：修饰及温敏诱导表达载体．生物化学与生物物理学报，1994，26：389-393．
[63] Studier F. W., Moffatt B. A.. Use of Bacteriophage T7 RNA Polymerase to direct selective high level expression of cloned genes. J. Mol. Biol., 1986, 189:113-118.
[64] Mcallister W.T., Carter A. D.. Regulation of promoter selection by the bacteriophage T7 RNA polymerase in vitro. Nucl Acids.Res., 1980, 8: 4821-4825.
[65] 萨姆布鲁克J．，弗里奇E．F．，曼尼阿蒂斯T．分子克隆实验指南．科学出版社，第一版，1986：19-20．
[66] 李德葆，周雪平，许建平等．基因工程操作技术．上海科学技术出版社，1988：27-28
[67] Sambrook J., MacCallum P., Russell D.. Molecular Cloning: A laboratory manual (3rd Edition). USA: Cold Spring Harbor Laboratory Press, 2001.
[68] Haaland P. D.. Experimental design in biotechnology. New York: ASQC Quality Press, 1989: 6.
[69] Plackett R. L., Burman J. P.. The design of optimum multifactorial experiments. Biometrika, 1946, 37: 305-325.
[70] Montgomery D. C.. Design and analysis of experiments (3rd ed.). USA: John Wiley & Sons, Inc., 1991: 521-568.
[71] Roquemore K. G.. Hybrid designs for quadratic response surfaces. Technometrics, 1976, 18(4): 419-423.
[72] Altamirano M. M., Garcia C., Possani L. D., Fersht A. R.. Oxidative refolding chromatography: folding of the scorpion toxin Cn5. Nature Biotechnology, 1999, 17: 187-191.