弱阳离子交换色谱固定相对还原变性溶菌酶的折叠贡献
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摘要
通过对8种不同种类弱阳离子交换色谱(WCX)填料对还原变性溶菌酶(Lys)复性的考察，选出了配基为3-硝基邻苯二甲酸酐柱为最佳复性的色谱柱，在此基础上，对WCX固定相对还原变性Lys折叠的贡献及其影响复性的各个条件进行研究。
全文包括四个部分：
1．文献综述：蛋白折叠的研究具有重大的理论意义和使用价值，液相色谱法(LC)是近年来发展最快的方法之一。本文仅对LC固定相对蛋白折叠的贡献以及二硫键折叠的研究进展进行了全面地综述。
2．为了减少不可逆吸附，在硅胶表面先键合了一层聚乙二醇(PEG)，以使硅胶表面变成亲水性，然后再对其表面进行化学改性使其成为弱阳离子交换(WCX)介质。亲水性的增强能够减少和避免生物大分子的不可逆吸附，并且可以维持其足够高的生物活性回收率。为了比较PEG对蛋白质复性的影响，本文用键合了不同分子量PEG的PEG200、PEG400、PEG600、PEG1000四种柱子，分别比较了它们对还原变性Lys复性的影响。结果表明从对还原变性Lys复性以及柱子稳定性方面考虑，键合PEG400的柱子效果最好。
3．由于在WCX中，在弱的离子交换剂上配体结构或组成的稍微变化就会对蛋白折叠产生较大的影响。研究了四种不同配基(3-硝基邻苯二甲酸酐、丁二酸酐、邻苯二甲酸酐、苯基丁二酸酐)的弱阳离子交换色谱填料对还原变性的Lys复性效率的影响。结果表明配基为3-硝基邻苯二甲酸酐的WCX柱复性效果最好。指出一个新概念，活性回收率(RB)与质量回收率(RM)之比R_((B／M))，通过实验检验，R_((B／M))可以很好的反映复性条件的好坏。
4．用WCX对还原变性Lys的复性进行了研究。考察了流动相中脲浓度及盐种类对用WCX对Lys复性的影响，结果表明当流动相中含有
3.Omol.L一’的脉及l.Omol·L一’硫酸钱时能提高其生物活性回收率。在此
wCX柱上，Lys的保留呈现离子交换和疏水双保留机理。发现在硅胶
基质的wex柱上，当蛋白浓度为1 5.omg·mL一’到so.omg·mL一’时，用
WCX折叠的复性产率均高于用稀释法的复性产率;在蛋白浓度为
20.omg’mL一’时活性回收率达到最高，为95.4%。同时还考察了流动相
组成、脉浓度、pH值、流速、温度和复性时间等对复性的影响。
The selecting of eight kinds of column of weak-cation exchange chromatography (WCX) was firstly investigated for the reduced/denatured lysozyme (Lys) refolding. The column bonding 3-nitrophthalic anhydride was found to be the best. The parameters effecting on the reduced/denatured Lys were also studied.
The thesis includes four parts as the follows:
1. Review: Protein folding is a subject of fundamental and practical importance and liquid chromatography (LC) is one of the new methods. Contribution of stationary phase to protein refolding and disulfide bond formation was reviewed, and 76 references were cited.
2. Using silica gel as matrix, many modified reactions to increase hydrophilicity were carried out by using polyethylene (PEG) with different molecular weights, such as PEG200, PEG400, PEG600, PEG 1000. The increases in the hydrophilicity of the stationary phase can reduce and avoid non-reversible adsorption of biopolymers. By evaluating the refolding of the reduced/denatured Lys with WCX column, PEG400 column was selected for the refolding of denatured/reduced Lys.
3. In WCX, a little changes of structure and constitute of ligands will cause a great effect on the Lys refolding. The four kinds of ligands, 3-nitrophthalic anhydride, succinic anhydride, phalic anhydride, and phenylsuccinic anhydride as the end groups were bond to the employed WCX column. Their contributions to the refolding of the denatured/reduced Lys were also evaluated, and the first one of them was found to be the best. A new definition called ratio R(b/m) of mass recovery (Rm) and bioactivity recovery (Rb) was firstly presented. With the R(b/m) the effect from experimental conditions of either adsorption or Lys refolding by LC can be judged
4. The refolding of the denatured/reduced Lys was investigated by using the WCX in the presence of the reduced and oxidized glutathione in the mobile phase employed. Effects of urea concentration and the kinds of salt on the renaturation of the reduced/denatured Lys were carefully studied. It was found that the Lys renaturation yield was significantly related to the urea concentration. When urea concentration was of 3.0molL-1 and (NH4)SO4 was as salt in the mobile phase, the bioactivity recovery of the renatured Lys was found to increase. The retention mechanism of the reduced Lys on the WCX column was proved to be a mixed mode of ion-exchange and hydrophobic interaction. With experimental optimization for Lys refolding, a considerably high bioactivity yield 95.38% was obtained, even though the initial concentration of Lys was raised up to 20.0 mg mL-1.
全文包括四个部分：
1．文献综述：蛋白折叠的研究具有重大的理论意义和使用价值，液相色谱法(LC)是近年来发展最快的方法之一。本文仅对LC固定相对蛋白折叠的贡献以及二硫键折叠的研究进展进行了全面地综述。
2．为了减少不可逆吸附，在硅胶表面先键合了一层聚乙二醇(PEG)，以使硅胶表面变成亲水性，然后再对其表面进行化学改性使其成为弱阳离子交换(WCX)介质。亲水性的增强能够减少和避免生物大分子的不可逆吸附，并且可以维持其足够高的生物活性回收率。为了比较PEG对蛋白质复性的影响，本文用键合了不同分子量PEG的PEG200、PEG400、PEG600、PEG1000四种柱子，分别比较了它们对还原变性Lys复性的影响。结果表明从对还原变性Lys复性以及柱子稳定性方面考虑，键合PEG400的柱子效果最好。
3．由于在WCX中，在弱的离子交换剂上配体结构或组成的稍微变化就会对蛋白折叠产生较大的影响。研究了四种不同配基(3-硝基邻苯二甲酸酐、丁二酸酐、邻苯二甲酸酐、苯基丁二酸酐)的弱阳离子交换色谱填料对还原变性的Lys复性效率的影响。结果表明配基为3-硝基邻苯二甲酸酐的WCX柱复性效果最好。指出一个新概念，活性回收率(RB)与质量回收率(RM)之比R_((B／M))，通过实验检验，R_((B／M))可以很好的反映复性条件的好坏。
4．用WCX对还原变性Lys的复性进行了研究。考察了流动相中脲浓度及盐种类对用WCX对Lys复性的影响，结果表明当流动相中含有
3.Omol.L一’的脉及l.Omol·L一’硫酸钱时能提高其生物活性回收率。在此
wCX柱上，Lys的保留呈现离子交换和疏水双保留机理。发现在硅胶
基质的wex柱上，当蛋白浓度为1 5.omg·mL一’到so.omg·mL一’时，用
WCX折叠的复性产率均高于用稀释法的复性产率;在蛋白浓度为
20.omg’mL一’时活性回收率达到最高，为95.4%。同时还考察了流动相
组成、脉浓度、pH值、流速、温度和复性时间等对复性的影响。
The selecting of eight kinds of column of weak-cation exchange chromatography (WCX) was firstly investigated for the reduced/denatured lysozyme (Lys) refolding. The column bonding 3-nitrophthalic anhydride was found to be the best. The parameters effecting on the reduced/denatured Lys were also studied.
The thesis includes four parts as the follows:
1. Review: Protein folding is a subject of fundamental and practical importance and liquid chromatography (LC) is one of the new methods. Contribution of stationary phase to protein refolding and disulfide bond formation was reviewed, and 76 references were cited.
2. Using silica gel as matrix, many modified reactions to increase hydrophilicity were carried out by using polyethylene (PEG) with different molecular weights, such as PEG200, PEG400, PEG600, PEG 1000. The increases in the hydrophilicity of the stationary phase can reduce and avoid non-reversible adsorption of biopolymers. By evaluating the refolding of the reduced/denatured Lys with WCX column, PEG400 column was selected for the refolding of denatured/reduced Lys.
3. In WCX, a little changes of structure and constitute of ligands will cause a great effect on the Lys refolding. The four kinds of ligands, 3-nitrophthalic anhydride, succinic anhydride, phalic anhydride, and phenylsuccinic anhydride as the end groups were bond to the employed WCX column. Their contributions to the refolding of the denatured/reduced Lys were also evaluated, and the first one of them was found to be the best. A new definition called ratio R(b/m) of mass recovery (Rm) and bioactivity recovery (Rb) was firstly presented. With the R(b/m) the effect from experimental conditions of either adsorption or Lys refolding by LC can be judged
4. The refolding of the denatured/reduced Lys was investigated by using the WCX in the presence of the reduced and oxidized glutathione in the mobile phase employed. Effects of urea concentration and the kinds of salt on the renaturation of the reduced/denatured Lys were carefully studied. It was found that the Lys renaturation yield was significantly related to the urea concentration. When urea concentration was of 3.0molL-1 and (NH4)SO4 was as salt in the mobile phase, the bioactivity recovery of the renatured Lys was found to increase. The retention mechanism of the reduced Lys on the WCX column was proved to be a mixed mode of ion-exchange and hydrophobic interaction. With experimental optimization for Lys refolding, a considerably high bioactivity yield 95.38% was obtained, even though the initial concentration of Lys was raised up to 20.0 mg mL-1.
引文
1. Anfinsen C B. [J] .Science. 1973,1981:223-230
2．孙彦，生物分离工程，北京：化学工业出版社，1998，23-34
3. Guise AD, West SM, Chaudhuri JB, Mol Biotechnol, 1996, 6:53-64
4. Chaudhuri JB. Ann NY Acad Sci,1994,721:374-385
5. Hagen A J, Hatton T A, Wang D I C, et al. Biotech Bioeng, 1990,35:955-965
6. Luisi P L, Giomini M, Pileni M P, et al. Biochem Acta, 1988,947:209-216
7. Creighton TE. Biochem Sci, 1997,22:6
8．耿信笃，常建华，李华儒等，高技术通讯，199l，1(7)：1-8
9. Geng X D, Chang X Q. J. Chromatogr., 1992,599:185-194
10. Suttnar J, Dyr J E, Hamsikova E, et al. J.Chromatogr. B,1994, 656(1):123-126
11. Werner M H, Clore G M, Groneborn A M, et al. FEBS Lett., 1994,345(23): 125-130
12. Taguchi H, Mokino Y, Yoshida M. J. Biol. Chem., 1994,269:8529-8634
13. Altamirano M M, Golbik R, Zahn R, et al. Proc Natl. Acad. Sci.,USA, 1997,94(8):3576-3578
14. Altamirano M M, Garcia C, Possani LD, et al. Nat. Biotechnol., 1999,17(2):187-191
15. Goldberg M E et al. Biochemistry, 1991,30:2790-2797
16．郭立安，高效液相色谱法纯化蛋白质理论与技术，陕西科学技术出版社，西安，1993
17．刘彤，耿信笃，色谱，2000，18(1)：30-35
18. Batas, B., Chaudhuri, J B., Biotechnol Bioeng., 1996,50:16-23
19. M H Werner, G. M Clore, A M Gronenborn, A Kondoh, R J Fisher, FEBS Letters, 1994,345:125-130
20. B Batas, J B Chaudhuri, Biotech.& Bioengineer., 1996,50(1):16-23
21. Muller, C., Rinas, U., J Chromatography A., 1999,855:203-213
22. Fahey, E M., Chaudhuri, J B., Binding, P., J Chromatography B., 2000,737:225-235
23. Werner M H, Clore G M, Groneborn A M,et al. FEBS Lett., 1994,345(23): 125-130
24. Rogl H, Kose mund K, Kuhlbrandt W et al. FEBS Lett, 1998,432(1-2):21-26
25. Zahn R, Von Schroetter C, Wuthrich K. FEBS Lett, 1997,417(3):400-404
26. Sinha D, Bakhshi M, Vora R. Bio Technique, 1994, 17:509-514
27. Yoshimoto M, Kuboi R, Yang Q et al. J Chromatogr B, 1998,712:59-71
28. Altamirano MM, Garcia C, Possani LD,et al. Nat Biotechnol, 1999,17(2): 187
29. 38.P K Jadhav, P J Ala, F J Woerner, et al., J Med Chem, 1997, 40(2), 181-191
30. P J Ala, E E Huston, R M Klabe, et al., Biochem., 1997, 1573-1580
31. P J Ala, R J Delossskey, E E Huston, et al., J Biol Chem.,1998: 273(20): 12325-12331
32. P J Ala, E E Huston, R M Klabe, et al., Biochem., 1998:15042-15049
33．白泉，孔宇，耿信笃，高等化学学报，2002，23(8)：1483-1488
34．白泉，孔宇，耿信笃，西北大学学报，2000，30：4851-4854
35．耿信笃，白泉，中国科学，2002，32(5)：460-471
36. Suttnar J, Dyr J E, Hamsikova E, et al. J. Chromatogr.B, 1994,656(1): 123-126
37. G Stempfer, B Holl-Neugebaur, R Rudolph, Nat. Biotech., 1996,14:329-334
38. T E Creighton, D L Oxender(eds), UCLA Symposia on Molecular and Cellular Biology, new series, 1986,39:249-257
39．沈同，王镜岩．生物化学．北京：高等教育出版社，1990．167
40．翟礼嘉，顾红雅，胡节，陈章良．现代生物技术导论．北京：高等教育出版社，施普林格出版社．1998．217
41. A S Acharya, H Taniuchi. Mol. Cell. Biochem. 1982, 44: 129-148
42. A S Acharya, H Taniuchi, J. Biol. Chem., 1976, 251: 6934-6946
43. A K Ahmed, S W Schaffer, D B Wetlaufer, J. Biol. Chem. 1975, 250:8477-8482
44. V P Saxena, D B Wetlaufer, Biochem., 1970, 9:5015-5022
45. C J Epstein, R A Goldberg, J. Biol. Chem. 1963, 238:1380-1383
46. C B Anfinsen, E Haber, J. Biol. Chem., 1961, 236: 1361-1363
47. T Isemura, T Takagi, Y Maeda, K Imai, Biochem. Biophys. Res. Comm., 1961, 5(5): 373-377
48. C T Duda, A Light, J. Biol. Chem., 1982, 257: 9866-9871
49. A Light, C T Duba, T W Odorzynski, W G I More, J. Cell. Biochem. 1986, 31: 19-26
50. T W Odorzynski, A Light. J Biol Chem, 1979, 254: 4294-4295
51. T E Creighton, Meth. Enzymol., 1986, 131: 83-106.
52. T E Creighton, D P Goldenberg, J. Mol. Biol, 1984, 179: 497-52
53. D M Rothwarf, H A Scheraga, J.Am. Chem,. Soc. 1991,113:6293-6294
54. N K Sinha, A Light, J. Biol. Chem., 1975, 250:8624-8629
55. D F Steiner, J I Clark, Proc. Natl. Acad. Sci. USA, 1969, 60:622-629
56．耿信笃，白泉，中国科学．2002．32(5)：460-471
57. Batas B, Chaudhuri J B.Biotechnol Bioeng, 1996,50:16-23
58. Batas B, Schiraldi C, Chaudhuri JB(1999).J Biotech 68:149-158
59. Fahey EM, Chaudhuri JB. J chromatogr B. 2000.737:225-235
60. Y Wang; B, L, Gong; X, Du, Geng. Chinese Chem. Lett. 2003, 14(8), 828-831
61．李敏，西北大学硕士学位论文，2003．5
62. L,M; Z,G,-F; Su,Z,-G. JChromatogr,2002, 959, 113-120
63．白泉．孔宇，张养军，耿信笃．西北大学学报(自然科学版)．2000．30(2)：127-130
64．王彦．耿信笃，色谱．2003，21(3)，218-221
65. R Zahn, C von Schroetter, K Wuthrich, FEBS Letters, 1997,417:400-404
66. M Yoshimoto, T Shimanouchi, H Umakoshi, R Kubio, J Chromatogr.B, 2000,743:93-99
67. M Yoshimoto, R Kubio, Biotechnol. Prog, 1999,15:480-487
68. M Yoshimoto, R Kubio, Q Yang, J Miyake, J. Chromatogr.B. 1998,712:59-71
69. Fleming A. Proc R Lond B Bio Sci, 1922,93:306
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76. Roux P, Delepierre M, Goldberg M E, Chaffotte A F. J Biochem, 1997,272(40):24 843
2．孙彦，生物分离工程，北京：化学工业出版社，1998，23-34
3. Guise AD, West SM, Chaudhuri JB, Mol Biotechnol, 1996, 6:53-64
4. Chaudhuri JB. Ann NY Acad Sci,1994,721:374-385
5. Hagen A J, Hatton T A, Wang D I C, et al. Biotech Bioeng, 1990,35:955-965
6. Luisi P L, Giomini M, Pileni M P, et al. Biochem Acta, 1988,947:209-216
7. Creighton TE. Biochem Sci, 1997,22:6
8．耿信笃，常建华，李华儒等，高技术通讯，199l，1(7)：1-8
9. Geng X D, Chang X Q. J. Chromatogr., 1992,599:185-194
10. Suttnar J, Dyr J E, Hamsikova E, et al. J.Chromatogr. B,1994, 656(1):123-126
11. Werner M H, Clore G M, Groneborn A M, et al. FEBS Lett., 1994,345(23): 125-130
12. Taguchi H, Mokino Y, Yoshida M. J. Biol. Chem., 1994,269:8529-8634
13. Altamirano M M, Golbik R, Zahn R, et al. Proc Natl. Acad. Sci.,USA, 1997,94(8):3576-3578
14. Altamirano M M, Garcia C, Possani LD, et al. Nat. Biotechnol., 1999,17(2):187-191
15. Goldberg M E et al. Biochemistry, 1991,30:2790-2797
16．郭立安，高效液相色谱法纯化蛋白质理论与技术，陕西科学技术出版社，西安，1993
17．刘彤，耿信笃，色谱，2000，18(1)：30-35
18. Batas, B., Chaudhuri, J B., Biotechnol Bioeng., 1996,50:16-23
19. M H Werner, G. M Clore, A M Gronenborn, A Kondoh, R J Fisher, FEBS Letters, 1994,345:125-130
20. B Batas, J B Chaudhuri, Biotech.& Bioengineer., 1996,50(1):16-23
21. Muller, C., Rinas, U., J Chromatography A., 1999,855:203-213
22. Fahey, E M., Chaudhuri, J B., Binding, P., J Chromatography B., 2000,737:225-235
23. Werner M H, Clore G M, Groneborn A M,et al. FEBS Lett., 1994,345(23): 125-130
24. Rogl H, Kose mund K, Kuhlbrandt W et al. FEBS Lett, 1998,432(1-2):21-26
25. Zahn R, Von Schroetter C, Wuthrich K. FEBS Lett, 1997,417(3):400-404
26. Sinha D, Bakhshi M, Vora R. Bio Technique, 1994, 17:509-514
27. Yoshimoto M, Kuboi R, Yang Q et al. J Chromatogr B, 1998,712:59-71
28. Altamirano MM, Garcia C, Possani LD,et al. Nat Biotechnol, 1999,17(2): 187
29. 38.P K Jadhav, P J Ala, F J Woerner, et al., J Med Chem, 1997, 40(2), 181-191
30. P J Ala, E E Huston, R M Klabe, et al., Biochem., 1997, 1573-1580
31. P J Ala, R J Delossskey, E E Huston, et al., J Biol Chem.,1998: 273(20): 12325-12331
32. P J Ala, E E Huston, R M Klabe, et al., Biochem., 1998:15042-15049
33．白泉，孔宇，耿信笃，高等化学学报，2002，23(8)：1483-1488
34．白泉，孔宇，耿信笃，西北大学学报，2000，30：4851-4854
35．耿信笃，白泉，中国科学，2002，32(5)：460-471
36. Suttnar J, Dyr J E, Hamsikova E, et al. J. Chromatogr.B, 1994,656(1): 123-126
37. G Stempfer, B Holl-Neugebaur, R Rudolph, Nat. Biotech., 1996,14:329-334
38. T E Creighton, D L Oxender(eds), UCLA Symposia on Molecular and Cellular Biology, new series, 1986,39:249-257
39．沈同，王镜岩．生物化学．北京：高等教育出版社，1990．167
40．翟礼嘉，顾红雅，胡节，陈章良．现代生物技术导论．北京：高等教育出版社，施普林格出版社．1998．217
41. A S Acharya, H Taniuchi. Mol. Cell. Biochem. 1982, 44: 129-148
42. A S Acharya, H Taniuchi, J. Biol. Chem., 1976, 251: 6934-6946
43. A K Ahmed, S W Schaffer, D B Wetlaufer, J. Biol. Chem. 1975, 250:8477-8482
44. V P Saxena, D B Wetlaufer, Biochem., 1970, 9:5015-5022
45. C J Epstein, R A Goldberg, J. Biol. Chem. 1963, 238:1380-1383
46. C B Anfinsen, E Haber, J. Biol. Chem., 1961, 236: 1361-1363
47. T Isemura, T Takagi, Y Maeda, K Imai, Biochem. Biophys. Res. Comm., 1961, 5(5): 373-377
48. C T Duda, A Light, J. Biol. Chem., 1982, 257: 9866-9871
49. A Light, C T Duba, T W Odorzynski, W G I More, J. Cell. Biochem. 1986, 31: 19-26
50. T W Odorzynski, A Light. J Biol Chem, 1979, 254: 4294-4295
51. T E Creighton, Meth. Enzymol., 1986, 131: 83-106.
52. T E Creighton, D P Goldenberg, J. Mol. Biol, 1984, 179: 497-52
53. D M Rothwarf, H A Scheraga, J.Am. Chem,. Soc. 1991,113:6293-6294
54. N K Sinha, A Light, J. Biol. Chem., 1975, 250:8624-8629
55. D F Steiner, J I Clark, Proc. Natl. Acad. Sci. USA, 1969, 60:622-629
56．耿信笃，白泉，中国科学．2002．32(5)：460-471
57. Batas B, Chaudhuri J B.Biotechnol Bioeng, 1996,50:16-23
58. Batas B, Schiraldi C, Chaudhuri JB(1999).J Biotech 68:149-158
59. Fahey EM, Chaudhuri JB. J chromatogr B. 2000.737:225-235
60. Y Wang; B, L, Gong; X, Du, Geng. Chinese Chem. Lett. 2003, 14(8), 828-831
61．李敏，西北大学硕士学位论文，2003．5
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