重组葡激酶的聚乙二醇选择性修饰
文献类型：学位论文
| 作者 | 王俊 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-11-24 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 |
| 关键词 | 固相修饰 聚乙二醇修饰 葡激酶 金属螯合 |
| 其他题名 | Studies on site-selective PEGylation for recombinant staphylokinase |
| 学位专业 | 生物化工 |
| 中文摘要 | 聚乙二醇修饰可以有效地降低蛋白质药物的免疫原性和抗原性，延长蛋白质药物在体内的循环半衰期。聚乙二醇修饰的长效蛋白质药物已成为近年来的研究热点。重组葡激酶是一种具有重要临床应用价值的蛋白质药物。本论文以开发高活性的长效葡激酶为目标，研究了聚乙二醇修饰重组葡激酶的反应动力学，利用金属螯合介质辅助聚乙二醇修饰带组氨酸标签（His-tag）的葡激酶，探索新的聚乙二醇定点修饰策略。本论文还建立了相应的聚乙二醇修饰工艺及修饰产物的分离纯化方法，获得了修饰位点均一且活性高的单修饰产物。聚乙二醇-丙醛是目前蛋白质修饰中应用非常广泛的一种修饰剂，它可以定点修饰蛋白质的N端，但也会修饰蛋白质的赖氨酸残基。本论文对聚乙二醇-丙醛修饰重组截长葡激酶的反应动力学进行了研究，考察了反应过程中修饰位点的变化规律，并检测了单修饰产物中N端和第121位赖氨酸的修饰率随反应时间的变化。研究发现在高修饰剂比条件下，N端和第121位赖氨酸的修饰率上升较快，但N端修饰所占比例较低。随着反应时间的延长，N端修饰所占比例逐渐增加。而在低修饰剂比条件下，虽然修饰率上升较慢，但在反应初期，单修饰产物中的N端修饰产物所占的比例就较高，基本达到定点修饰。反应动力学的研究结果可以指导反应过程的控制，有利于制备出修饰位点均一的单修饰产物。根据全长葡激酶的特殊溶栓机理，用聚乙二醇-丙醛修饰全长葡激酶的N端，根据之前反应动力学的研究，对反应过程进行控制，制备出位点均一的单修饰产物，即Mono-PEG5K-SAK和Mono-PEG20K-SAK。这两种单修饰产物保留了原蛋白100%的生物活性。研究还发现经PEG修饰后，全长葡激酶与纤溶酶结合并切除葡激酶N端10个肽段的特性没有改变，但切除速率会随着偶联PEG链分子量的增大而减小。修饰产物的热稳定性和抗胰蛋白酶解能力比原蛋白均有显著提高。利用金属螯合介质来辅助琥珀酰亚胺碳酸酯-单甲氧基聚乙二醇（SC-mPEG）修饰带组氨酸标签的葡激酶。首先制备了6种不同配基密度和孔径大小的金属螯合介质，从中筛选出修饰率最高的介质进行后续的固相修饰研究。实验结果表明与液相修饰相比， SC-mPEG进行固相吸附修饰时的反应可控性有很大提高。等电聚焦电泳分析结果表明，固相修饰得到的单修饰产物比液相修饰的单修饰产物位点更均一。胰蛋白酶酶解图谱和生物活性检测结果表明，由于固相修饰时葡激酶的活性中心区域因被介质吸附而避免被聚乙二醇修饰，固相修饰产物的生物活性要高于液相修饰产物。分子量为5、10和20 kDa的SC-mPEG修饰剂在液相修饰中所得单修饰产物的生物活性分别是原蛋白的78.6%、62.5%和58%；固相修饰所得单修饰产物的生物活性分别是原蛋白的84.3%、82.5%和78%。本研究还把蛋白的分离纯化及聚乙二醇修饰反应耦合到一步反应中，简化了操作步骤。 |
| 英文摘要 | PEGylation, i.e., covalent attachment of polyethylene glycol （PEG） to proteins, is an effective method to improve the therapeutic potentials of proteins by prolonging their in vivo half-life, decreasing their immunogenicity and increasing their pharmaceutical properties. In our study, the systemic PEGylation studies on recombinant staphylokinase （SAK） were conducted via selection of PEG reactant and PEGylation method. The corresponding modification technics and purification technology were established and the homogeneous mono-PEGylated SAKs with relatively high activities were obtained. In addition, using the specific mechanism of dissolve thrombus, a kind of controlled-release PEGylated SAK was prepared. Conjugation with polyethylene glycol (PEGylation) specifically at the N terminus of proteins using aldehyde chemistry has been widely used to improve the pharmacological profiles of proteins. Due to the presence of the competitive PEGylation between N terminus and the lysine residues, analysis of the N-terminal PEGylation process is of significant importance. Staphylokinase (SAK) has high fibrin selectivity for thrombolytic therapy of acute myocardial infarction and is used as the model protein in the present work. Thus, kinetic and stoichiometric study on the PEGylation of SAK was carried out. In the PEGylation process, the PEGylation extent of SAK molecule and its amino acid residues (e.g., Met1 and Lys121) were analyzed using size exclusion chromatography and tryptic peptide mapping, respectively. The mono-PEGylated SAK (mono-PEG-SAK) was obtained with a maximal yield of ~60% at the PEG to SAK molar ratio of 10:1 at 3-5 h and pH 5.0, consisting of the positional isomers at Met1 and Lys121. However, the mono-PEG-SAK was essentially N-terminal PEG-SAK (i.e., at Met1) with a yield of ~50% at 8 h, along with the presence of ~50% di-PEG-SAK. In contrast, the mono-PEG-SAK was essentially the N-terminal PEG-SAK at the PEG to SAK molar ratio of 4:1 during the PEGylation process (i.e., 2-8 h). The mono-PEG-SAK has a yield of ~28% at 8 h and pH 5.0, along with the presence of ~72% unmodified SAK and the absence of the di-PEG-SAK. Thus, our present study can provide the optimal conditions for N-terminal PEGylation of proteins by analysis of the N-terminal PEGylation process. We used PEG-ALD5K and PEG-ALD20K to modify the N-terminus of rSAK and obtained mono-PEG5K-rSAK and mono-PEG20K-rSAK, respectively. These two products showed peculiarity of controlled release when reacted with plasmin and remained the full bioactivity of unmodified rSAK. Then we investigated the solid-phase PEGylation using immobilized metal-ion medium. Firstly, six kinds of medium with different apertures and densities of Ni2+ were synthesized. The Ni2+-IDA-Sepharose 4FF (15 mmol) exhibited the highest PEGylation degree and was thus selected for the solid-phase PEGylation. After sonication of the bacteria, supernatant containing recombinant staphylokinase was loaded onto the IMAC media. Sequentially, succinimidyl carbonate mPEG （5, 10 and 20 kDa） were introduced to the media and allowed to react with the bounded staphylokinase. The mono-PEG-SAK obtained by this method showed almost the same characters with that from liquid phase when assayed by circular dichroism, intrinsic fluorescence, trypsin resistance and thermal stability. However, isoelectric focusing electrophoresis analysis indicated that mono-PEG-SAKs obtained from solid phase PEGylation showed only one band while those from liquid phase PEGylation displayed two bands. Furthermore, tryptic peptide mapping showed that the crucial bioactive position of N terminus was completely protected during the PEGylation procedure in solid-phase PEGylation, thereby resulting in a much higher bioactivity. |
| 语种 | 中文 |
| 公开日期 | 2013-09-24 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1756]  |
| 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 | 王俊. 重组葡激酶的聚乙二醇选择性修饰[D]. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：过程工程研究所
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