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题名	离子交换色谱复性重组人粒细胞集落剌激固子的研究
作者	罗天乐
学位类别	硕士
答辩日期	2004
授予单位	中国科学院过程工程研究所
授予地点	中国科学院过程工程研究所
导师	苏志国
关键词	重组人粒细胞集落刺激因子 离子交换色谱 复性 精氨酸 反相色谱
其他题名	Study on the Refolding of Recombinant Human Granulocyte-Colony Stimulating Factor by Ion Exchange Chromatography
学位专业	生物化工
中文摘要	本文研究了以包含体形式表达的重组人粒细胞集落刺激因子（rhG-CSF）在常用的 脉一精氨酸辅助复性溶液体系中的复性过程，在稀释复性的基础上，着重考察了离子 交换色谱复性，并以反相色谱为主要分析手段探讨了rhG-CSF在两种复性方式中折 叠的不同特点，探讨了rhG-CSF在不同复性方式下复性效果不同的原因，分析了影 响离子交换色谱复性效果的主要因素并提出了改进的思路。首先考察并比较了rhG-CSF在无添加剂的缓冲液、加有L-精氨酸的缓冲液、以 及脉一精氨酸混合溶液中的稀释复性的特点。发现了用脉作为变性系统的rhG-CSF包 含体蛋白稀释复性的蛋白收率要明显高于用盐酸肌作变性剂的现象，并发现在服一精 氨酸混合溶液中稀释时，复性体系中的脉浓度对于折叠平衡状态的影响很大，必须低 于4mol／L刁一能使rhG-CSF复性。 然后研究了以DEAE SePharose Fast Flow为代表的阴离子交换柱上复性 rhG-CSF。除了使用常规的NaCl溶液作为洗脱剂外，尝试了将精氨酸直接用作离子 交换色谱复性的洗脱剂，显著地提高了色谱复性效率。通过比较三缓冲液系统（流动 相按高浓度变性剂一低盐复性缓冲液一高盐复性缓冲液的顺序转换）和双缓冲液系统（流动相按高浓度变性剂一高盐复性缓冲液的顺序转换）的两种洗脱方式对于复性效 果的影响，提出：1）在被吸附于离子交换介质上的状态下，rhG-CSF难以折叠复性， 复性在洗脱过程中，蛋白具有一定的空间自由度时才能发生，因此，通过降低蛋白质 分子数和色谱吸附位点数的比值来减小洗脱时蛋白在柱中的拥挤程度可能是提高复 性效率的一个重要途径；2）洗脱时流动相中足够低的腮浓度对于rhG-CSF复性是重 要的，双缓冲液系统洗脱方式复性的rhG-CSF的活性之所以比稀释复性和三缓冲液 系统洗脱方式复性的rhG-CSF都明显偏低，和洗脱时流动相脉浓度太高有关。此外，还尝试了在CM Sepharose Fast Flow阳离子交换柱上复性rhG-CSF，将合 适的pH梯度与脉梯度联合使用可以明显提高复性效果，但阳离子交换复性的pH难 以控制，容易生成无活性的中间体，总蛋白收率也比较低。用反相色谱作为主要分析手段对rhG-CSF在精氨酸溶液中，脉一精氨酸混合溶液 中的稀释复性，阴离子交换色谱柱上的穿过复性以及阴、阳离子交换色谱复性进行了 监测，比较了它们各自的特点，并由此对各种复性方法的复性效果差异进行了解释，发现并着重分析了阴离子交换色谱复性的rhG-CSF独特的折叠状态。实验中还发现，如果将阴离子交换色谱复性的rhG-CSF进一步稀释，则其RPC 图谱特征将向稀释复性的特征转化，而且其活性增高。这提示将离子交换色谱复性和 稀释复性结合起来，利用稀释手段弥补色谱复性的不足是有可能的。
英文摘要	In this thesis, ion exchange chromatography(IEC) was utilized as a primary tool to refold recombinant human granulocyte colony-stimulating factor(rhG-CSF) expressed in Escherichia coli as inclusion body in urea-arginine solutions. Comparisons were made of the behaviors of rhG-CSF in dilution refolding and in various modes of IEC refolding by means of reversed phase chromatography(RPC). Factors influencing the efficacy of IEC refolding were analyzed and considerations for solution were proposed. Firstly, the conventional refolding by dilution was studied. The denatured rhG-CSF from inclusion body was diluted into additive-free buffers, buffers containing 0.5mol/L L-arginine, and buffers containing a series of concenctrations of urea and L-arginine and the results were compared. An intriguing fact was observed that substituting urea for guanidine hydrochloride as the solvent of the rhG-CSF inclusion body extract, either by solubilizing the inclusion body directly with urea or by desalting the GdnHCl-dissolved rhG-CSF inclusion body extract with urea, substantially reduced aggregation in subsequent dilution as measured by gross protein recovery. The urea concentration in the refolding buffer was proved decisive to refolding equilibrium: urea concentrations above 4mol/L in the refolding buffer forbade full renaturation of rhG-CSF. Refolding by anion exchange chromatography(A-IEC) was then intensively investigated, promarily on DEAE Sepharose Fast Flow column. L-arginine, typically an addictive used in dilution, was introduced to the A-IEC refolding as an eluent in addition to the commonly used NaCl. Two modes of elution: the tribuffer mode and dibuffer mode, were compared. In the tribuffer mode, the denatured rhG-CSF was absorbed onto the anion exchange medium under high concentration of denaturant(buffer A), then allowed to refold while buffer A was replaced with refolding buffer(buffer B), and then washed off the medium by the eluting buffer(buffer C); while in the dibuffer mode, buffer A was directly converted to buffer B which doubled as refolding buffer and eluting buffer. Based on the results it was proposed that refolding of the rhG-CSF molecules cannot take place when they are chained to the medium, but in the elution process, and consequently reducing the crowding of attached rhG-CSFs might be conducive to refolding yield; and that urea concentration should be low enough in the course of elution to ensure proper refolding, which may explain why rhG-CSF refolded by dibuffer-mode A-IEC had much lower activity than those refolded by dilution or tribuffer-mode A-IEC. On a tentative basis, refolding was conducted using cation exchange chromagtography(C-IEC). A pH-urea dual gradient was applied to the C-IEC and the specific activity of the refolded rhG-CSF rose significantly, which may be attributed to the suppression of disulfide misparing by low pH. However, refolding on cation exchanger often resulted in low protein recovery and some stable, inactive intermediates. To probe the possible difference in refolding patterns of rhG-CSF in dilution and IEC-based refolding processes, RPC was employed to monitor and charaterize the behavior of rhG-CSF in dilution, flow-through refolding(rhG-CSF passing through an A-IEC column perfused with refolding buffer without attaching to it), and A-IEC refolding in both tribuffer and dibuffer modes, as well as in C-IEC refolding on CM Fast Flow column, and based on the result explanations were given for the difference in refolding efficacy of those refolding procedures. It was demonstrated that rhG-CSF refolded by A-IEC was in a unique state marked by asynchronous refolding kenetics and conformational variability. Another discovery was that dilution of rhG-CSF refolded by IEC could and its activity increased evidently, indicating the feasibility of combining dilition and IEC refolding to overcome the problems confronted in IEC refolding.
语种	中文
公开日期	2013-09-16
页码	79
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1433]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	罗天乐. 离子交换色谱复性重组人粒细胞集落剌激固子的研究[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004.

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