疏水色谱法对猪心中细胞色素C的分离纯化及液相色谱法对还原变性牛胰岛素折叠特性的研究
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摘要
液相色谱不仅是生物大分子分离纯化的有效手段，同时也是一种研究变性蛋白质折叠的有力手段。本论文主要包括两个部分，首先使用简易型疏水色谱柱对猪心中的细胞色素C进行了纯化分离，获得了99％以上的纯度，98％以上的质量回收率；另外，使用体积排阻色谱，疏水色谱，离子交换色谱法对还原变性的牛胰岛素的再折叠特性进行了研究。全文共包括以下五个章节：
1．文献综述 对液相色谱的基本理论---计量置换保留理论和短柱理论作了简要阐述，并介绍了一种本实验室依据以上理论研制的简易型色谱柱及其应用；对蛋白质复性中的一些基本理论及方法进行了阐述和比较，说明了液相色谱作为一种新的复性手段其基本原理和优点，并简要介绍了胰岛素复性研究的意义。
2．疏水色谱法从猪心中分离纯化细胞色素C 提出了一种从猪心中快速分离纯化和制备高纯度细胞色素C(Cyt-C)的新方法。使用一种装有大颗粒疏水填料的简易型疏水色谱柱对猪心中的Cyt-C进行了分离纯化，并通过反相色谱，SDS-聚丙烯酰胺凝胶电泳，MALDI-TOF MS，紫外图谱，及铁含量对样品的纯度进行了检验。与经典方法相比，此方法操作时间短，纯化工艺简单，质量回收率和纯度可分别提高至98.5％和99％以上。另外，从柱子放大，规模扩大等方面进行了初步研究，为将此方法用于制备规模的研究奠定了基础。所使用的简易型色谱柱具有价格便宜，分离效率高，柱寿命长等优点，具有十分广泛的应用价值。
3．体积排阻色谱法对还原变性牛胰岛素折叠特性的研究 用体积排阻色谱法(SEC)对还原变性牛胰岛素(Ins)的再折叠特性进行了研究。分别考察了使用不同流动相及流动相中添加GSSG／GSH及不同浓度脲对还原变性Ins折叠效率的影响。结果表明，还原变性Ins在含有GSSG／GSH及2.0 mol·L~(-1)脲浓度的流动相中复性效果最好。此外，使用脲梯度法对还原变性Ins进行了折叠研究。通过改变脲梯度时间对还原变性Ins折叠行为进行了研究，结果表明采用12mL脲梯度最适宜，对还原变性Ins的复性效果最好。上述结果用
反相色谱法进一步进行了验证。对稀释法、SEC法及脉梯度SEC法对还原脉
变、肌变InS的复性结果进行了比较，结果表明脉梯度法对还原变性Ins的复
性效果最好，SEC法次之，稀释法最差，且还原腮变Ins复性效果要好于还
原肌变Ins。
4.疏水色谱法对还原变性牛胰岛素折叠特性的研究用疏水色谱法(HIC)对
还原变性牛胰岛素的再折叠特性进行了研究。分别考察了在HIC流动相中改
变GSSG/GSH对比率及腮浓度对还原变性InS折叠效率的影响。结果表明流
动相中添加GSSG/GSH比率为6:1及脉浓度为Zmol·L’’时对还原变性Ins的
复性效果最好，并用反相色谱对这一结果进行了进一步的验证。对稀释法与
HIC法对还原变性his的复性效果进行了比较，结果表明HIC法对还原变性
Ins的复性效果优于稀释法。
5.离子交换色谱法对还原变性牛胰岛素折叠特性的研究用强阴离子交换色谱
法对还原变性胰岛素的再折叠特性进行了研究。分别考察了IEC流动相中盐
种类，添加GSSG/GSH对，不同服浓度及pH值对还原变性Ins折叠效率的
影响。结果表明在流动相中添加GssG/GsH及2.0 mol·L一1脉且pH值为8时
对还原变性InS的复性效果最好。通过紫外吸收光谱和荧光光谱对还原变性
Ins的复性结果进行检验，进一步证实了流动相中添加GSSG/G SH对和2.0
mol·L一‘脉有利于还原变性Ins的复性和二硫键的正确对接。对稀释法与IEC
法对还原变性Ins的复性效果进行了比较，并用紫外吸收光谱和荧光光谱加
以检验，结果表明IEC法的复性效果优于稀释法。
Liquid chromatography (LC) is one of the most effective way to separate and purify biopolymer, and is also a powerful tool to be used for protein refolding. This thesis includes two parts: Firstly the purification and separation of cytochrome-c (Cyt-C) from pig heart with simple type hydrophobic interaction chromatography (HIC) column was investigated, and the purity and mass recovery of Cyt-C can be obtained to be 99% and 98.5%, respectively. Secondly the refolding of reduced-denatured insulin from bovine pancreas with size-exclusion chromatography (SEC), HIC, and ion-exchange chromatography (IEC) was also investigated, respectively. This thesis includes five chapters as follows:
1.Review: The fundamental theories of LC - stoichiometric displacement theory for retention (SDT-R) and short column theory were briefly introduced, and a new type of column -simple-type column which was designed in our laboratory based on the two theories described above was also introduced in this chapter. Then some of the basic theories and methods of protein refolding were presented and compared, and the significance of the refolding of insulin was also illuminated.
2. Purification and separation of Cyt-C from pig heart by HIC. A fast method for separation and purification of Cyt-C of high purity from pig heart is presented. A simple-type column of hydrophobic interaction chromatography packed with big particles was employed for the fast purification. The purity of the purified Cyt-C was tested by reversed phase liquid chromatography (RPLC), SDS-PAGE, MALDI-TOF MS, UV spectrum, and iron content in it. Compared with the common method, the new method has advantages of spending short time, having simpler purification processes, and high mass recoveries of 98.5%, high purity of 99%. In addition, the enlarging of the column and scale was investigated primarily. The simple column used has the advantages of low prices, good separate efficiency, long-time life etc., which can be applied broadly.
3. Investigation on the refolding of the reduced-denatured insulin with SEC.
The reduced-denatured insulin from bovine pancreas denatured with 7.0 mol.L-1 guanidine hydrochloride (GuHCl) and 8.0 mol.L-1 urea, respectively, was refolded by using SEC. The effects of different mobile phases and concentration of urea on chromatographic behavior of reduced-denatured insulin with SEC were investigated. The results indicated that the mobile phase containing GSSG/GSH and 2.0 mol.L-1 urea was the best condition to refold insulin with SEC. These results were tested with RPLC further. In addition, the urea gradient SEC was used to refold the reduced-denatured insulin. The results showed that urea gradient SEC was also a good way to enhance the refolding efficiency of reduced -denatured insulin. Comparing the refolding efficiency of reduced-denatured insulin with dilution method, SEC and urea gradient SEC and testing the results with RPLC, it was found that SEC for protein refolding is much more advantageous than dilution method, and urea gradient SEC can be used to improve refolding efficiency of insulin more than common method. The refolding efficiency of urea reduced-denatured insulin refolded with SEC is more than GuHCl reduced-denatured insulin.
4. Investigation on the refolding of the reduced-denatured insulin with HIC. The reduced-denatured insulin denatured with 7.0 mol.L-1 GuHCl and 8.0 mol.L-1 urea, respectively, was refolded by using HIC. The effects of different concentration of GSH/GSSG and urea on chromatographic behavior of reduced -denatured insulin with HIC were investigated. The results indicated that the mobile phase with GSSG/GSH of 6:1 and 2.0mol.L urea was the best condition to refold insulin with HIC, and with the increasing of the concentration of urea in the mobile phase, the retention time of the proteins was decreased. With the comparison of the refolding efficiency of insulin refolded by the dilution method and HIC, it was found that HIC is much more advantageous to be used for refolding the reduced-den
1．文献综述 对液相色谱的基本理论---计量置换保留理论和短柱理论作了简要阐述，并介绍了一种本实验室依据以上理论研制的简易型色谱柱及其应用；对蛋白质复性中的一些基本理论及方法进行了阐述和比较，说明了液相色谱作为一种新的复性手段其基本原理和优点，并简要介绍了胰岛素复性研究的意义。
2．疏水色谱法从猪心中分离纯化细胞色素C 提出了一种从猪心中快速分离纯化和制备高纯度细胞色素C(Cyt-C)的新方法。使用一种装有大颗粒疏水填料的简易型疏水色谱柱对猪心中的Cyt-C进行了分离纯化，并通过反相色谱，SDS-聚丙烯酰胺凝胶电泳，MALDI-TOF MS，紫外图谱，及铁含量对样品的纯度进行了检验。与经典方法相比，此方法操作时间短，纯化工艺简单，质量回收率和纯度可分别提高至98.5％和99％以上。另外，从柱子放大，规模扩大等方面进行了初步研究，为将此方法用于制备规模的研究奠定了基础。所使用的简易型色谱柱具有价格便宜，分离效率高，柱寿命长等优点，具有十分广泛的应用价值。
3．体积排阻色谱法对还原变性牛胰岛素折叠特性的研究 用体积排阻色谱法(SEC)对还原变性牛胰岛素(Ins)的再折叠特性进行了研究。分别考察了使用不同流动相及流动相中添加GSSG／GSH及不同浓度脲对还原变性Ins折叠效率的影响。结果表明，还原变性Ins在含有GSSG／GSH及2.0 mol·L~(-1)脲浓度的流动相中复性效果最好。此外，使用脲梯度法对还原变性Ins进行了折叠研究。通过改变脲梯度时间对还原变性Ins折叠行为进行了研究，结果表明采用12mL脲梯度最适宜，对还原变性Ins的复性效果最好。上述结果用
反相色谱法进一步进行了验证。对稀释法、SEC法及脉梯度SEC法对还原脉
变、肌变InS的复性结果进行了比较，结果表明脉梯度法对还原变性Ins的复
性效果最好，SEC法次之，稀释法最差，且还原腮变Ins复性效果要好于还
原肌变Ins。
4.疏水色谱法对还原变性牛胰岛素折叠特性的研究用疏水色谱法(HIC)对
还原变性牛胰岛素的再折叠特性进行了研究。分别考察了在HIC流动相中改
变GSSG/GSH对比率及腮浓度对还原变性InS折叠效率的影响。结果表明流
动相中添加GSSG/GSH比率为6:1及脉浓度为Zmol·L’’时对还原变性Ins的
复性效果最好，并用反相色谱对这一结果进行了进一步的验证。对稀释法与
HIC法对还原变性his的复性效果进行了比较，结果表明HIC法对还原变性
Ins的复性效果优于稀释法。
5.离子交换色谱法对还原变性牛胰岛素折叠特性的研究用强阴离子交换色谱
法对还原变性胰岛素的再折叠特性进行了研究。分别考察了IEC流动相中盐
种类，添加GSSG/GSH对，不同服浓度及pH值对还原变性Ins折叠效率的
影响。结果表明在流动相中添加GssG/GsH及2.0 mol·L一1脉且pH值为8时
对还原变性InS的复性效果最好。通过紫外吸收光谱和荧光光谱对还原变性
Ins的复性结果进行检验，进一步证实了流动相中添加GSSG/G SH对和2.0
mol·L一‘脉有利于还原变性Ins的复性和二硫键的正确对接。对稀释法与IEC
法对还原变性Ins的复性效果进行了比较，并用紫外吸收光谱和荧光光谱加
以检验，结果表明IEC法的复性效果优于稀释法。
Liquid chromatography (LC) is one of the most effective way to separate and purify biopolymer, and is also a powerful tool to be used for protein refolding. This thesis includes two parts: Firstly the purification and separation of cytochrome-c (Cyt-C) from pig heart with simple type hydrophobic interaction chromatography (HIC) column was investigated, and the purity and mass recovery of Cyt-C can be obtained to be 99% and 98.5%, respectively. Secondly the refolding of reduced-denatured insulin from bovine pancreas with size-exclusion chromatography (SEC), HIC, and ion-exchange chromatography (IEC) was also investigated, respectively. This thesis includes five chapters as follows:
1.Review: The fundamental theories of LC - stoichiometric displacement theory for retention (SDT-R) and short column theory were briefly introduced, and a new type of column -simple-type column which was designed in our laboratory based on the two theories described above was also introduced in this chapter. Then some of the basic theories and methods of protein refolding were presented and compared, and the significance of the refolding of insulin was also illuminated.
2. Purification and separation of Cyt-C from pig heart by HIC. A fast method for separation and purification of Cyt-C of high purity from pig heart is presented. A simple-type column of hydrophobic interaction chromatography packed with big particles was employed for the fast purification. The purity of the purified Cyt-C was tested by reversed phase liquid chromatography (RPLC), SDS-PAGE, MALDI-TOF MS, UV spectrum, and iron content in it. Compared with the common method, the new method has advantages of spending short time, having simpler purification processes, and high mass recoveries of 98.5%, high purity of 99%. In addition, the enlarging of the column and scale was investigated primarily. The simple column used has the advantages of low prices, good separate efficiency, long-time life etc., which can be applied broadly.
3. Investigation on the refolding of the reduced-denatured insulin with SEC.
The reduced-denatured insulin from bovine pancreas denatured with 7.0 mol.L-1 guanidine hydrochloride (GuHCl) and 8.0 mol.L-1 urea, respectively, was refolded by using SEC. The effects of different mobile phases and concentration of urea on chromatographic behavior of reduced-denatured insulin with SEC were investigated. The results indicated that the mobile phase containing GSSG/GSH and 2.0 mol.L-1 urea was the best condition to refold insulin with SEC. These results were tested with RPLC further. In addition, the urea gradient SEC was used to refold the reduced-denatured insulin. The results showed that urea gradient SEC was also a good way to enhance the refolding efficiency of reduced -denatured insulin. Comparing the refolding efficiency of reduced-denatured insulin with dilution method, SEC and urea gradient SEC and testing the results with RPLC, it was found that SEC for protein refolding is much more advantageous than dilution method, and urea gradient SEC can be used to improve refolding efficiency of insulin more than common method. The refolding efficiency of urea reduced-denatured insulin refolded with SEC is more than GuHCl reduced-denatured insulin.
4. Investigation on the refolding of the reduced-denatured insulin with HIC. The reduced-denatured insulin denatured with 7.0 mol.L-1 GuHCl and 8.0 mol.L-1 urea, respectively, was refolded by using HIC. The effects of different concentration of GSH/GSSG and urea on chromatographic behavior of reduced -denatured insulin with HIC were investigated. The results indicated that the mobile phase with GSSG/GSH of 6:1 and 2.0mol.L urea was the best condition to refold insulin with HIC, and with the increasing of the concentration of urea in the mobile phase, the retention time of the proteins was decreased. With the comparison of the refolding efficiency of insulin refolded by the dilution method and HIC, it was found that HIC is much more advantageous to be used for refolding the reduced-den
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