摘要
ApoE是一种阿朴质蛋白(apolipoprotein),它有299个氨基酸。它可以同LDL受体蛋白、肝素以及磷酯相结合,其主要的生理功能是调节体内磷酯的代谢。它同第三类高酯血、动脉硬化及老年痴呆病相关。了解apoE的结构以及其在体内的分子生物学过程,可提高人类对这些疾病的预防以及治疗的水平。ApoE氨基端(1-199)的结构已用X-Ray法进行了测定,而apoE羧酸端及整个分子的结构依然未知。由于apoE蛋白质分子量较大,NMR信号重叠较多,不利于结构的解析以及片段相互作用的研究。我们拟利用INTEIN媒介蛋白质拼接方法,制备部分同位素标记的apoE ,降低NMR结构解析的难度。受体相关蛋白(Receptor-associated protein, RAP)是一种LDL受体折叠的伴侣蛋白,它由323个氨基酸组成,同apoE一样,可以同LDL受体蛋白结合,两者之间有一定的相似性。本文着重研究了利用INTEIN媒介蛋白质拼接法制备apoE,并对RAP及各片段性质进行了初步的研究。
在使用蛋白质拼接法制备部分同位素标记apoE的过程中,详细研究了相关蛋白的制备及拼接反应的条件,产物的纯化。在制备羧酸端的过程中,使用三种不同的方法:直接法制备,使用CNBr切断法以及凝血因子Xa酶切方法,经比较发现凝血因子Xa酶切法无论时收率还是稳定性方面均较成功,但该方法的缺点是存在非特异性的酶切位点;在对apoE氨基端研究过程中,发现使用碱性的培养介质可提高蛋白质的表达收率,使用简单含巯基的化合物进行硫醇解后所得产物会发生水解,无法用于下步的反应;在比较了不同的蛋白质拼接方法后,发现使用INTEIN媒介的蛋白质拼接法可以得到较好的效果;使用优化后的反应条件,第一次大量地制备了部分片段同位素标记的apoE,并进行了NMR测定,得到了简化了的NMR谱图。为进一步使用NMR研究apoE的结构提供了可能。本方法对于制备其它部分同位素标记的蛋白质有借鉴意义。
针对在使用pTYB1表达载体表达蛋白质的过程中出现的自裂解现象,进行了比较分析,有针对性提出的解决方案。所得到的两种变异体可以部分或几乎完全禁阻自裂解。所使用的方法具有通用性,完全可应于其它的蛋白质的表达过程。
使用蛋白质交联反应,脂结合能力测定及CD等手段,对RAP及其片段的热力学性质进行了初步研究。
ApoE is one kind of apolipoprotein of 299 amino acids. It can bind to LDL receptor protein, lipid and heparin. It plays important roles in modulating the metabolism of lipid and associated to the type III hyperlipoproteinemia, atherosclerosis, and Alzheimer’s disease (AD). The understanding of the structure and molecular biological function of apoE is important and will help us to prevent and cure those diseases. The structure of apoE N-terminal (apoE1-199) has been solved with X-Ray, apoE C-terminal and full-length apoE are still unknown. ApoE is too big to solve the structure by NMR, so we try to prepare segmental isotopic labeling apoE for NMR study. Receptor-associated protein (RAP) is a chaperon protein of LDL receptor protein, it is consist of 323 amino acid residues. It can bind to the immature LDL receptor and help it refold correctly. In this paper, we study the methodology of preparing the full-length apoE by protein ligation and some property of full length RAP and some domain of RAP.
In this paper, different ways were tried to prepare the alpha-Cys C-terminal, alpha-thioester N-terminal and carry out ligation. Three ways were tried to prepare alpha-Cys c-terminal: directly to expression, CNBr and Factor was use to cut fusion protein. After introducing reducing agent BME, factor Xa cleavage was found suitable to achieve this goal. To prepare alpha-thioester, apoE N-terminal was subcloning into IMPACT expression system. It is first time to observe that the high yield of protein can be gotten from basic medium, it also be found most protein contain alpha-thioester after thiolysis are easily hydrolysis to carboxyl acid which can’t be used in protein ligation. Different ways were tried to carry out the protein ligation, Intein-mediated protein ligation was found to be the best way to get ligation product. Finally CL-6B heparin column was used to purify to product. Segmental isotopic labeling apoE were prepared in large scale for NMR study.
In order to block the autocleavage of fusion protein in pTWIN1 vector during expression, two mutants were successfully design base on protein engineering. RAP was also studied with cross-linking, CD and lipid binding assay.
引文
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