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Alternative TitleMolecular insight into the steric shielding effect of PEG on the conjugated staphylokinase
Thesis Advisor余景开 ; 胡涛
Degree Grantor中国科学院研究生院
Degree Discipline生物工程
Keyword聚乙二醇化   葡激酶   分子模拟   生物活性   空间位阻
Abstract随着蛋白质化学和分子生物学的迅速发展,用于各种疾病治疗的蛋白质(酶)类药物的研制与应用已经成为生物医药产业发展的热点。而药用蛋白质的抗原性、半衰期等因素已成为制约蛋白质药物发展的关键因素。聚乙二醇化可以提高蛋白质药物在体内的循环半衰期以及降低免疫原性和抗原性,因此成为改善蛋白药效的一种有效手段。然而,PEG修饰会造成蛋白质药物出现明显的生物活性的损失,原因可能是PEG链对蛋白的空间位阻效应以及PEG链对蛋白环境的微观影响。 本论文以葡激酶(Sak)为模型蛋白,它具有很好的溶栓效应,但其体内循环半衰期很短,通过PEG化可以明显延长它的半衰期,同时带来生物活性的损失。为了更好地揭示PEG修饰对蛋白药物的影响,本研究分别考察修饰位点及修饰链长对蛋白药物的结构、活性影响,并通过分子模拟和对接分析从分子水平上揭示PEG在蛋白表面的构象和PEG修饰对蛋白及其底物间结合能的影响。本论文的主要成果在于: 首先,系统的考察PEG修饰位点及链长对蛋白Sak的结构、活性影响。分别在Sak的N末端和C末端进行定点修饰,链长分别选取分子量5 kDa和20 kDa。结构鉴定结果发现与Sak相比,PEGylated-Sak的二级结构和三级结构发生了轻微的改变。体外活性实验显示活性损失与PEG链长成正比,且与修饰位点相关,相同链长情况下N端活性损失比C端活性损失大。 其次,分子模拟研究揭示了PEG对蛋白的影响主要是由于PEG链包裹在蛋白表面形成的空间屏蔽效应。PEG在蛋白表面形成水化层,水化层的厚度与PEG链长近似呈正比,并且与修饰位点相关。修饰样品与底物纤溶酶原的对接分析揭示了不同位点、链长修饰时结合能的变化。 计算模拟的结果与实验数据达到了很好的一致性(R2>0.98)。模拟结果揭示了PEG在蛋白表面的构象以及PEG对Sak和其底物结合力的影响。本研究中实验与模拟相结合的手段可以帮助进行理性的蛋白药物设计。
Other AbstractWith advances in protein chemistry and molecular biology, research and application of proteins/enzymes for therapeutical purposes had become a major topic in the biomedical industry. However, many protein drugs have shortcoming such as high antigenicity, short serum half-life et al., which slowed development of effective protein drugs. PEGylation is conjugation of polyethylene glycol (PEG) to a therapeutic protein. Proper PEGylation can increase serum half-life, decrease immunogenicity and antigenicity thus become an effective approach for improving therapeutic potency of protein drugs. Unfortunately, PEGylation can lead to apparent loss of biological activity. Steric shielding effect of PEG chain and its influence on protein microenvironment are possible factors causing bioactivity reduction. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, with a very short serum half-life, was used as a model protein in this research. PEGylation increases the half-life of Sak, accompanied by loss of bioactivity. We investigated the effect of PEGylation site and PEG chain length on the structure and activity of Sak. Molecular dynamics (MD) simulation and docking analyses were used to reveal atomic level PEG conformation on protein surface and effect of PEGylation on Sak's ability to bind its substrate. The present study achieved the following results. Firstly, PEG effect on structure and activity of protein was systematically evaluated with deferent PEGylation sites and chain lengths. The N-terminus and C-terminus of Sak were chosen to carry out site-specific PEGylation. PEG chain of 5 kDa and 20 kDa were chosen. We found that the secondary and tertiary structure of protein had very little changes during PEGylation. The in vitro bioactivity was inversely proportional to the PEG chain length and dependent on PEGylation site. N-terminal PEGylation induced higher bioactivity loss compared with the C-terminal PEGylation. Secondly, molecular dynamics simulation suggested that the steric shielding effect of PEG wrapping at the surface of protein was the main factor afffecting protein bioactivity. The hydration layer was formed and its thickness was proportional to the chain length and different at two sites. Docking analyses indicated that the binding affinity of Sak to its receptor, plasminogen, is dependent on the PEG chain length and the PEGylation site. Computational and experimental data are found to correlate well (R2>0.98) with each other, which shows that experimental data support simulation results and simulation data are able to clarify PEG conformation on protein surface and PEG effect on Sak's ability to bind its substrate. The type of hybrid computational and experimental approach presented here can aid rational protein drug design projects.
Document Type学位论文
Recommended Citation
GB/T 7714
穆启蒙. 聚乙二醇修饰重组葡激酶中空间位阻的分子模拟研究[D]. 中国科学院研究生院,2013.
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