还原变性核糖核酸酶的液相色谱折叠法研究
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摘要
本文利用计量置换参数Z表征了不同变性态溶菌酶在弱阳离子交换色谱上的构象变化规律。在此基础上对还原变性核糖核酸酶在离子交换色谱(IEC)，疏水色谱(HIC)及排阻色谱(SEC)中的复性进行研究。
全文包括五个部分：
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，本文对蛋白复性的基本策略，及在这些基本策略下发展出的各种复性方法及进展进行了全面地综述。
2．以计量置换理论(stoichiometric displacement theory for retention，SDT-R)为基础，以溶菌酶(Lys)为目标蛋白，用SDT-R中的参数Z和logI对弱阳离子交换色谱(WCX)中“准天然态”和脲变还原与非还原两种变性状态Lys的分子构象变化进行了表征。发现在流动相中含有脲时，蛋白的保留仍服从SDT-R理论，可准确测定在该特定脲浓度条件下Lys的Z及logI值。结果表明，3种分子构象状态下的Z值均随脲浓度的改变呈现不连续变化；“准天然态”Lys在不同脲浓度条件下的Z值比变性状态的大，logI比变性状态的小，而非还原变性态和准天然态的Z和logI值比较接近。还对不同脲浓度条件下Lys的Z值与活性回收率之间的关系进行了研究。
3．用WCX对还原变性的核糖核酸酶A(RNase A)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复性RNase A的影响，结果表明当流动相中含有1.0-2.0mol/L的脲时能提高其生物活性回收率。在此WCX柱上，天然RNase A的保留呈现离子交换和疏水双保留机理。研究了固定相对WCX复性RNase A的影响，比较了以硅胶基质，凝胶基质及聚合物基质三种WCX固定相填料对于RNase A复性的影响，其有各自的优点。发现在硅胶基质的WCX柱上，蛋白浓度高达30.00mg/mL时，可获得活性回收率达84.70％的复性效果。同时考察了流动相组成，脲浓度、pH值、流速和复性时间等对复性的影响。
4．研究了还原变性RNase A在疏水色谱(HIC)上的复性情况。在蛋白浓度为
3.omg/mL时可以达到80%以上，但在更高的蛋白浓度时其复性效率显著降低。
还考察了流动相组成，脉浓度、pH值、流速和复性时间等对复性的影响。
5.研究了还原变性RNaseA在尺寸排阻色谱(SEC)上的复性情况，认为SEC是一
种较好的色谱复性方法。氧化型和还原型谷肤甘肚的加入对复性有积极作用，
同时加入适当浓度的脉是必不可少的。脉梯度复性是一种对用SEC对蛋白复
性法的改进，它在色谱过程中线性脱除变性剂崛，为蛋白质的再折叠提供了一
个较温和的环境，但流速的快慢对其复性的最终效果并没有大的影响。
The stoichiometric displacement parameters Z value of Lys in different molecular states were investigated firstly and RNase A refolding by ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) was studied.
The thesis includes five parts as the follows:
1. Review: Protein folding is a subject of fundamental and practical importance. A fundamental refolding strategy was introduced. All kinds of methods and development based on the strategy were reviewed.
2. Based on the stoichiometric displacement theory for retention (SDT-R), the parameters Z and log/ were used to characterize the molecular conformational changes of lysozyme (Lys) for different molecular conformation states (pseudo-native, urea-unfolded, urea-reduced-unfolded) under various urea concentrations in weak cation exchange chromatography (WCX). The retention of the three molecular conformations of Lys totally follow the SDT-R .The Z values of the Lys in its pseudo-native state decreased with the increasing the concentration of urea in the mobile phase and was the biggest of the three molecular conformational states, while its corresponding log/ was the least one. Both Z and log/ of the Lys in their pseudo-native and urea-unfolded states were closed with each other. The changes of Z of Lys in the three molecular conformational states with urea concentration in mobile phase were found to be
discontinuously. The relationship between the Z value and bioactivity recovery of Lys under different urea concentrations was also investigated.
3. Oxidative refolding of the denatured/reduced Ribonuclease A (RNase A) was investigated by using weak cation exchange chromatography (WCX) with the presence of reduced and oxidized glutathione in the mobile phase employed. Effects of urea concentration and the kind of salt on the renaturation of
reduced/denatured RNase A were investigated. It was found that the renaturation
yield was significantly related to the urea concentration, urea concentration of 1.0-2.0mol/L in the mobile phase was found to increase the bioactivity recovery. The retention mechanism of the reduced RNase A on the WCX column was proved to be a mixed mode of ion-exchange and hydrophobic interaction. With the comparison of three kinds of stationary phase of WCX with the different matrixes, such as silica, sepharose and polymer, it was found every one had its own advantage. With experimental optimization for RNase A refolding, a considerably high bioactivity yield 84.7% was obtained even though the initial concentration of RNase A was raised up to 30.0 mg/ml by using of silica matrix column. The effects of the composition of mobile phase, urea concentration, pH, flow rate and the refolding time on the RNase A refolding were investigated.
4. Reduced /denatured RNase A was refolded by using hydrophobic interaction chromatography (HIC). The highest bioactivity yield, 86.4% was obtained from the weekly hydrophobicity of HIC column, when the loading RNase A concentration was 3.0mg/mL. The bioactivity yield decreased with the increase of the loading protein concentration. The contribution of the composition of the mobile phase, urea concentration, pH, flow rate, and refolding time to RNase A refolding were also investigated .
5. Refolding of reduced/denatured RNase A was compared by using size exclusion chromatography (SEC). In the presence of reduced and oxidized glutathione in mobile phase is necessary. With a linear decreased urea gradient urea gradient SEC also enhanced the yield of protein refolding, it provided a suitable environment to RNase A refolding .The flow rate of mobile phase was not found to have any important effects on the bioactivity yield of RNase A.
全文包括五个部分：
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，本文对蛋白复性的基本策略，及在这些基本策略下发展出的各种复性方法及进展进行了全面地综述。
2．以计量置换理论(stoichiometric displacement theory for retention，SDT-R)为基础，以溶菌酶(Lys)为目标蛋白，用SDT-R中的参数Z和logI对弱阳离子交换色谱(WCX)中“准天然态”和脲变还原与非还原两种变性状态Lys的分子构象变化进行了表征。发现在流动相中含有脲时，蛋白的保留仍服从SDT-R理论，可准确测定在该特定脲浓度条件下Lys的Z及logI值。结果表明，3种分子构象状态下的Z值均随脲浓度的改变呈现不连续变化；“准天然态”Lys在不同脲浓度条件下的Z值比变性状态的大，logI比变性状态的小，而非还原变性态和准天然态的Z和logI值比较接近。还对不同脲浓度条件下Lys的Z值与活性回收率之间的关系进行了研究。
3．用WCX对还原变性的核糖核酸酶A(RNase A)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复性RNase A的影响，结果表明当流动相中含有1.0-2.0mol/L的脲时能提高其生物活性回收率。在此WCX柱上，天然RNase A的保留呈现离子交换和疏水双保留机理。研究了固定相对WCX复性RNase A的影响，比较了以硅胶基质，凝胶基质及聚合物基质三种WCX固定相填料对于RNase A复性的影响，其有各自的优点。发现在硅胶基质的WCX柱上，蛋白浓度高达30.00mg/mL时，可获得活性回收率达84.70％的复性效果。同时考察了流动相组成，脲浓度、pH值、流速和复性时间等对复性的影响。
4．研究了还原变性RNase A在疏水色谱(HIC)上的复性情况。在蛋白浓度为
3.omg/mL时可以达到80%以上，但在更高的蛋白浓度时其复性效率显著降低。
还考察了流动相组成，脉浓度、pH值、流速和复性时间等对复性的影响。
5.研究了还原变性RNaseA在尺寸排阻色谱(SEC)上的复性情况，认为SEC是一
种较好的色谱复性方法。氧化型和还原型谷肤甘肚的加入对复性有积极作用，
同时加入适当浓度的脉是必不可少的。脉梯度复性是一种对用SEC对蛋白复
性法的改进，它在色谱过程中线性脱除变性剂崛，为蛋白质的再折叠提供了一
个较温和的环境，但流速的快慢对其复性的最终效果并没有大的影响。
The stoichiometric displacement parameters Z value of Lys in different molecular states were investigated firstly and RNase A refolding by ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) was studied.
The thesis includes five parts as the follows:
1. Review: Protein folding is a subject of fundamental and practical importance. A fundamental refolding strategy was introduced. All kinds of methods and development based on the strategy were reviewed.
2. Based on the stoichiometric displacement theory for retention (SDT-R), the parameters Z and log/ were used to characterize the molecular conformational changes of lysozyme (Lys) for different molecular conformation states (pseudo-native, urea-unfolded, urea-reduced-unfolded) under various urea concentrations in weak cation exchange chromatography (WCX). The retention of the three molecular conformations of Lys totally follow the SDT-R .The Z values of the Lys in its pseudo-native state decreased with the increasing the concentration of urea in the mobile phase and was the biggest of the three molecular conformational states, while its corresponding log/ was the least one. Both Z and log/ of the Lys in their pseudo-native and urea-unfolded states were closed with each other. The changes of Z of Lys in the three molecular conformational states with urea concentration in mobile phase were found to be
discontinuously. The relationship between the Z value and bioactivity recovery of Lys under different urea concentrations was also investigated.
3. Oxidative refolding of the denatured/reduced Ribonuclease A (RNase A) was investigated by using weak cation exchange chromatography (WCX) with the presence of reduced and oxidized glutathione in the mobile phase employed. Effects of urea concentration and the kind of salt on the renaturation of
reduced/denatured RNase A were investigated. It was found that the renaturation
yield was significantly related to the urea concentration, urea concentration of 1.0-2.0mol/L in the mobile phase was found to increase the bioactivity recovery. The retention mechanism of the reduced RNase A on the WCX column was proved to be a mixed mode of ion-exchange and hydrophobic interaction. With the comparison of three kinds of stationary phase of WCX with the different matrixes, such as silica, sepharose and polymer, it was found every one had its own advantage. With experimental optimization for RNase A refolding, a considerably high bioactivity yield 84.7% was obtained even though the initial concentration of RNase A was raised up to 30.0 mg/ml by using of silica matrix column. The effects of the composition of mobile phase, urea concentration, pH, flow rate and the refolding time on the RNase A refolding were investigated.
4. Reduced /denatured RNase A was refolded by using hydrophobic interaction chromatography (HIC). The highest bioactivity yield, 86.4% was obtained from the weekly hydrophobicity of HIC column, when the loading RNase A concentration was 3.0mg/mL. The bioactivity yield decreased with the increase of the loading protein concentration. The contribution of the composition of the mobile phase, urea concentration, pH, flow rate, and refolding time to RNase A refolding were also investigated .
5. Refolding of reduced/denatured RNase A was compared by using size exclusion chromatography (SEC). In the presence of reduced and oxidized glutathione in mobile phase is necessary. With a linear decreased urea gradient urea gradient SEC also enhanced the yield of protein refolding, it provided a suitable environment to RNase A refolding .The flow rate of mobile phase was not found to have any important effects on the bioactivity yield of RNase A.
引文
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2. F A O Marston, Biochem. J., 1986,240:1-12
3. A Mitraki, J King, Bio/Technol.1989, 7:690-697
4. A Mukhopadhyay, Adv. Biochem. Eng. Biotechnol., 1997, 56:61-109
5. T Kohno, D F Carmichael, A Sommer, et al, Methods Enzymol., 1990, 185:187-195
6． 时亚丽，马风，耿信笃．高等学校化学学报，1994，15(9)：1288—1291
7． 时亚丽，马风，耿信笃．分析化学，1994，22(7)：712—715
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14. Y Xie, D B Wetlaufer, Protein Sci., 1996, 5:517-523
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