| 题名 | 蛋白质的超滤复性和小分子伴侣辅助复性的机理 |
| 作者 | 赵大伟 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 |
| 关键词 | 超滤复性 精氨酸 氢氘交换 等温滴定量热 粒细胞集落刺激因子 |
| 其他题名 | Ultrafiltration refolding and the mechanism of small molecular chaperone assistance in protein reofolding |
| 学位专业 | 生物化工 |
| 中文摘要 | 体外复性是重组蛋白质药物生产过程中的重要操作单元。根据复性过程改变复性溶液的微环境是提高包涵体蛋白质体外复性效率的关键。本文以重组人粒细胞集落刺激因子（rhG-CSF）为模型，利用超滤技术提高包涵体蛋白质的体外复性浓度及复性收率。同时，采用氢氘交换液质联用及等温滴定微量热技术对小分子添加剂辅助蛋白质复性的机理进行了研究。 稀释复性的策略在rhG-CSF高浓度复性时仅能获得低的复性收率，复性动力学分析表明其原因rhG-CSF的中间体的形成及转化均为慢反应过程。超滤操作方式可以避免传统稀释复性过程中变性剂浓度急剧降低的情况，为蛋白质复性提供了温和变化的溶液环境，通过控制变性剂的去除速率使其和蛋白质的复性速率相匹配。反向高效液相色谱分析显示，超滤复性过程有效促进了变性态结构及复性中间体向正确折叠结构的转化；圆二色和荧光光谱分析表明蛋白质的二、三级结构在超滤过程中得以恢复。在蛋白质浓度为1.0 mg/mL时，超滤复性的可溶蛋白质收率仍然可以维持在51%，复性蛋白质的比活达到了1.51×108 IU/mg，分别是相同条件下稀释复性的1.5倍和3.3倍。在此基础上，将亲水性高分子聚合物引入超滤复性过程中，实验结果证实超滤复性的效率得到进一步改善。PEG在超滤复性提供的逐渐降低的变性剂环境中可以有效的和复性中间体相互作用，使其完全转化为正确折叠结构，复性蛋白质的比活提高至2.30×108 IU/mg；dextran则通过减少超滤过程中剪切率对蛋白质的破坏作用使可溶性蛋白质的收率增加至64%。超滤复性样品经过一步阳离子交换层析即可获得纯度大于99%的目标蛋白，结构及活性分析表明其与标准品具有相同的性质。此外，纯化操作的时间大幅削减为原来的1/6。 作为低分子量复性辅助剂的代表，精氨酸常用于蛋白质体外复性过程以提高复性收率，但其作用机理一直未阐述清楚。本文利用氢氘交换质谱和等温滴定量热技术对精氨酸在rhG-CSF复性过程中的作用机理进行研究，弥补了传统研究复性机理的方法仅能提供复性过程中蛋白质整体结构变化信息的不足，为揭示其作用机制提供了详细且直观的依据。rhG-CSF复性过程中，当精氨酸存在时蛋白质碳骨架上的酰胺氘原子的交换反应速率明显降低，表明精氨酸与蛋白质之间存在相互作用。结合对交换反应产物的胃蛋白酶酶解分析，可以进一步确定rhG-CSF分子中16-47、72-84、84-93、114-124、145-153和154-162六个肽段是蛋白质与精氨酸发生结合反应的重要区域。通过作用区域的氨基酸序列分析及蛋白质晶体结构所提供的信息，推断精氨酸通过静电作用、疏水作用及氢键与蛋白质结合。等温滴定量热技术提供的热力学数据显示精氨酸和变性态蛋白质之间存在结合作用，而和天然态蛋白质之间无相互作用。综合以上结果，推断精氨酸在蛋白质复性过程中的作用机理为：通过多种类型作用方式与变性态及部分复性态蛋白质结合，减少了蛋白质分子间相互作用导致的聚集体的形成，随着蛋白质结构的恢复，精氨酸逐渐从蛋白质分子上剥离出来，从而并不会影响蛋白质结构的转化，当蛋白质恢复其天然态结构后，精氨酸和蛋白质之间便不再有相互作用。 |
| 英文摘要 | In vitro refolding is the most important operation unit in the production process of recombinant protein drugs. Adjustment of the micro-environment of the refolding solution to match protein refolding process is crucial for improving the refolding efficiency of inclusion bodies. In this paper, selecting recombinant human granulocyte-colony stimulating factor (rhG-CSF) as the model protein, ultrafiltration was applied to increase the refolding concentration and the refolding recovery. Furthermore, the mechanism of small molecule additive assisting protein refolding was investigated by using HDX-MS and ITC technology. Dilution refolding strategy only provided low refolding yield at high rhG-CSF concentration, which mainly resulted form the slow rate of the formation and transformation of refolding intermediate. During ultrafiltration process, denaturant concentration decreases gradually, providing a modest changed environment for protein refolding. Furthermore, the removal rate of denaturant could be controlled to better match protein refolding rate. RP-HPLC analysis showed that ultrafiltration refolding effectively promoted the transformation of denatured protein and refolding intermediate into the native state. Results of circular dichroism and intrinsic fluorescence spectroscopy confirmed that the secondary and tertiary structure of rhG-CSF revived in ultrafiltration refolding process. Soluble protein recovery and specific activity of rhG-CSF for ultrafiltration refolding at protein concentration of 1.0 mg/mL can still be maintained at 51% and 1.51×108 IU/mg which were 1.5-fold and 3.3-fold higher than those for dilution refolding under the same condition. On this basis, hydrophilic polymers were introduced into this process to further improve the refolding efficiency. Combining with ultrafiltration, hydrophilic polymers embodied different mechanism. PEG could interact with the refolding intermediates effectively in the gradually changed environment and assisted it transformed into the correctly folded conformation. With the assistance of PEG, specific activity of rhG-CSF for ultrafiltration refolding increased to 2.30×108 IU/mg. As for dextran, it could increase the soluble protein recovery to 64% by protecting protein from being damaged by shear stress. rhG-CSF could be obtained with more than 99% purity by one-step cation-exchange chromatography. Structure and bioactivity of purified rhG-CSF were identical to those of the standard protein. Furthermore, time consumption reduced to one sixth for protein purification compaired with that for dilution refolding. As a low molecualr chaperone, arginine is commonly used to improve protein refolding, however, the mechanism of its assistance in protein refolding has not been clearly elucidated. In this dissertation, hydrogen/deuterium exchange mass spectrum and isothermo titration calorimetry experiments were performed to investigate the effects of arginine in protein refolding and provid more direct information to disclose the mechanism of arginine action. During rhG-CSF refolding process, HDX/MS analysis revealed that the exchange rate of the amide deuterim atoms significantly reduced in the presence of arginine indicating the interaction between protein and arginine. Combined with pepsin digestion of exchanged protein, six peptides spaning 16-47, 72-84, 84-93, 114-124, 145-153 and 154-162 of rhG-CSF were identified as the regions which contributed more to the combination between protein and arginine. Integrating amino acid sequence analysis of those peptides and protein crystal structure, it can be deduced that arginine combines with protein via a multi-type interaction including electrostatic interaction, hydrophobic interaction and hydrogen bonding. Thermodynamic data obtained from ITC experiments showed that arginine interact with denatured protein but not with the native one. Getting all the results together, the hypothesis of the mechanism of arginine assistance in protein |
| 语种 | 中文 |
| 公开日期 | 2015-07-08 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/15513]  |
| 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 | 赵大伟. 蛋白质的超滤复性和小分子伴侣辅助复性的机理[D]. 中国科学院研究生院. 2014. |
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