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题名	精氨酸辅助蛋白质折叠机理研究及过程优化
作者	陈净
学位类别	博士
答辩日期	2009-04-16
授予单位	中国科学院过程工程研究所
授予地点	过程工程研究所
导师	苏志国
关键词	蛋白质复性 精氨酸 疏水聚集 共价聚集 层析复性
其他题名	The mechanism investigation and process optimization of arginine-assisted refolding
学位专业	生物化工
中文摘要	精氨酸是一种在蛋白质折叠复性中应用最广泛的聚集抑制剂，但是研究发现精氨酸对于某些含二硫键蛋白的聚集几乎没有抑制作用，目前对于其作用机理还未做过系统研究。本文选取了结构性质完全不同的CAB、溶菌酶、GFP和rhG-CSF为模型蛋白，考察了精氨酸辅助蛋白质折叠的过程特征，深入研究了精氨酸辅助蛋白质折叠的机理，为蛋白质折叠的过程控制和优化提供了理论基础。 结果表明，精氨酸能有效抑制蛋白质分子间的疏水聚集，但是不能抑制由巯基错配形成的共价聚集，这是精氨酸对某些含二硫键蛋白的聚集没有抑制作用的主要原因。其具体表现为，不含二硫键的CAB在0.75 M以上精氨酸溶液中的蛋白收率能达到95%以上，且可溶性蛋白全部以单体形式存在；含有一个游离巯基的rhG-CSF即使是在高浓度的精氨酸溶液中复性时，都出现了大量共价交联的不可溶性聚集，但用碘乙酰胺把rhG-CSF的五个巯基封闭后，在0.5 M的精氨酸溶液中复性的蛋白则全部以可溶性形式存在；同时对含有一对二硫键的GFP和四对二硫键的溶菌酶的复性过程研究发现，高浓度精氨酸会提高由共价结合形成的可溶性聚集体。复性动力学研究表明造成可溶性共价聚集体增多的原因是由于高浓度的精氨酸能够减慢蛋白质的折叠速度，从而增加了含有游离巯基折叠中间体的接触机会。 针对精氨酸不能抑制共价聚集及减慢折叠速度的缺陷，以rhG-CSF为模型蛋白，分别尝试了精氨酸与折叠促进剂（甘油和蔗糖）、氧化还原体系（GSH/GSSG，β-巯基乙醇）及聚集抑制剂（盐酸胍和脲）之间的协同作用，发现精氨酸和脲组成的协同体系能够大幅度提高折叠效率。当采用0.5 M精氨酸和2 M脲组成的协同体系复性时，能将rhG-CSF的活性收率由40%提高至80%以上，并且该协同体系能够克服文献中rhG-CSF不能在高浓度下复性的不足，在蛋白终浓度为0.425 mg/ml时，活性收率还能达到65%。凝胶过滤，SDS-PAGE和复性动力学分析表明，该协同体系的作用机理为：脲和精氨酸虽然都能抑制疏水聚集，但脲主要是抑制了不可溶的疏水聚集，对可溶性的疏水聚集几乎没有抑制作用，而精氨酸的作用正好相反，主要是抑制了疏水作用形成的可溶性聚集，二者在功能上具有互补关系，通过协同作用加强对疏水作用的抑制，从而减少了折叠中间体之间的疏水区域靠近，因此也就无法进一步结合形成共价聚集。 将0.5 M精氨酸和2 M脲的协同作用体系与凝胶过滤层析、离子交换层析以及疏水层析相结合复性rhG-CSF，能够进一步减少聚集，提高活性收率。在凝胶过滤层析柱顶端引入0.3 CV的脲梯度后再进样，活性收率为70%时，得到的复性蛋白终浓度为0.67 mg/ml，是在稀释复性法中得到相同活性收率的蛋白终浓度的3倍，实现了rhG-CSF在高浓度下的复性；在双缓冲液系统离子交换层析复性中引入精氨酸和脲，复性蛋白终浓度为0.32 mg/ml，活性收率达到了80%以上，比同等条件下的稀释复性提高了10%。在协同作用体系辅助的疏水层析复性rhG-CSF过程中，由于rhG-CSF与介质之间的强吸附，最高活性收率仅为50%，但加入脲和精氨酸，仍能够提高rhG-CSF在被测的四种介质上的复性收率。而在疏水层析复性中换用疏水性较弱的溶菌酶时，脲和精氨酸组成的协同作用体系能够有效的将其蛋白收率提高至89.5%。 将精氨酸作为固定相，以2 M脲作为流动相是本文新发展起来的复性系统。采用自制的溴化氰活化精氨酸琼脂糖凝胶 6FF柱复性溶菌酶，在配基密度为56 μmol/g湿介质时，流速为0.5 ml/min的复性效率最高，蛋白收率达到96%，活性收率达到85%；而复性包涵体变性蛋白rhG-CSF时，流速0.2 ml/min复性效率最高，在蛋白终浓度为0.562 mg/ml时仍保留了53.4%的活性收率。
英文摘要	Among all the refolding additives, arginine is one of the most frequently employed to suppress aggregation during protein refolding. However, arginine does not work for certain disulfide-bond-containing proteins, which is not yet well explained. In the present work, refolding results of CAB, lysozyme, GFP and rhG-CSF, in the presence of arginine, were investigated and compared. Our results showed that arginine could effectively suppress hydrophobic interaction, but was of no effect on covalent binding. CAB, containing no cysteine, was successfully refolded with the help of arginine. The protein yield could reach 95% in the presence of 0.75 M arginine. However, rhG-CSF, containing five cysteines, could only achieve 65% protein yield. The formation of aggregates was found. Blocking of free SH groups of the denatured rhG-CSF by iodoacetamide and subsequently refolding of the protein could reduce the insoluble aggregate formation substantially. Further investigation on GFP and lysozyme refolding revealed arginine even promoted covalent oligomers. And analysis of the refolding kinetics indicated that covalent oligomers were probably formed in the intermediate stage where arginine slowed down the refolding rate and stabilized the intermediates. The accumulated intermediates with unpaired cysteine possessed more chances to react with each other to form oligomers, whereas arginine failed to inhibit disulfide bond formation. To overcome the limitation of arginine-assisted refolding, we investigated the combined use of arginine with “folding enhancer” (glycerol and sucrose), redox system (GSH/GSSG, β-ME) or “aggregation inhibitors” (GdmCl and urea), respectively. And a cooperative system composed of 2 M urea and 0.5 M arginine was proved to effectively improve rhG-CSF refolding. This cooperative system could increase the activity yield from 40% to 80%. And after optimization, the monomer yield kept 65% even in final protein concentration at as high as 0.425 mg/ml. By SEC, SDS-PAGE and refolding kinetics analysis, the mechanism of cooperative system was revealed. Although both urea and arginine could inhibit hydrophobic aggregates, urea could inhibit insoluble hydrophobic aggregates effectively but not soluble hydrophobic oligomers, on the contrary, arginine was more effective in inhibiting soluble hydrophobic oligomers than insoluble aggregates, suggesting a complementary effect existed in cooperative system composed of urea and arginine. Therefore, the suppression of hydrophobic interaction by this system could decrease “proximity effect” and further lessen the chances of forming covalent aggregates. In the process of SEC, IEC and HIC refolding of rhG-CSF, using 0.5 M arginin and 2 M urea as mobile phase could further increase refolding yields. The monomer yield reached 68% at the final protein concentration of 0.67 mg/ml in SEC refolding and 80% at 0.32 mg/ml in IEC refolding, which was much better than dilution refolding. Urea and arginine also could improve the protein yields of HIC refolding, but the hydrophobicity of rhG-CSF was too strong to get high yields, only 50%. However, for lysozyme with weak hydrophobicity, the monomer yield reached 89.5%. A new refolding system was developed by immobilizing arginine to cyanogen bromide activated Sehparose 6FF and using 2 M urea as the mobile phase. The highest ligand density was 56 μmol/g wet media. The protein yield of lysozyme was 96% with 85% of activity yield when refolded on this arginine column at a flow rate of 0.5 ml/min. And for rhG-CSF refolding, the monomer yield was 53.4% with final protein concentration of 0.562 mg/ml at a flow rate of 0.2 ml/min.
语种	中文
公开日期	2013-09-13
页码	157
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1121]
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	陈净. 精氨酸辅助蛋白质折叠机理研究及过程优化[D]. 过程工程研究所. 中国科学院过程工程研究所. 2009.

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