囊泡转运及重大疾病相关膜蛋白的纯化方法与蛋白性质研究
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摘要
囊泡转运和融合过程作为细胞生长繁殖的基本特征之一,在蛋白上膜及分泌途径中发挥重要的功能。囊泡的转运和分泌在各个阶段都需要一系列具有特殊功能的蛋白质的参与。Munc13-1在细胞中囊泡的启动分泌阶段发挥着重要作用。Exocyst复合物在囊泡的拴系和锚定过程中同样不可或缺。目前,对此类蛋白的性质和特征以及蛋白的三维晶体结构的研究有利于我们深入了解它们在囊泡转运过程中的分子作用机制。本文采用原核表达体系和层析技术表达纯化了一系列具有结构功能特征的Munc13-1缺失突变体,结合圆二色光谱,动态光散射和分析超速离心等体外蛋白研究技术对各缺失突变体蛋白的性质进行了系统的分析和比较。Munc13-1各缺失体蛋白的聚合状态,颗粒均一性及蛋白稳定性都存在着显著的差异。根据圆二色光谱和分析超速离心实验结果,我们首次发现了Munc13-1蛋白在溶液中的长棒状伸展螺旋束构象结构。结合同源建模构建了Munc13-1蛋白在体内以单体方式发挥功能的模型。Exocyst复合物的亚基蛋白Sec10的体外表达纯化研究发现,Sec10蛋白本身作为一个相对折叠紧密的结构来发挥功能,蛋白N端或者C端氨基酸的部分缺失都不可避免的导致了Sec10在表达过程中的错误折叠,形成包涵体。电镜分析发现了聚合的Sec10蛋白大部分以球形结构存在。
GABAB受体属于G蛋白偶联受体中的C蛋白家族,与各类神经和精神错乱疾病有关。我们通过低温表达和包涵体变性复性实验得到了可用于结晶的具有药物结合靶点活性的胞外蛋白片断。葡萄糖转运蛋白GLUT4(Glucose Transporter 4)作为II型糖尿病领域研究中的关键蛋白,在维持体内糖平衡起到重要作用。目前世界上还未有关于体外组织大规模纯化GLUT4的相关报道。我们首次尝试选用猪脂肪作为原材料,采用组织匀浆,差速离心和硫酸铵沉淀方法初步分离纯化并获得了足够蛋白产量和纯度的GLUT4。
膜蛋白的纯化与结晶是当前结构生物学领域里的一大难题。当前蛋白表达提取,蛋白性质优化等研究为进一步的结晶学研究打下基础。
The process of vesicle translocation and fusion, as a ubiquitous and fundamental event in the cell growth and propagation, play a critical role in proteins trafficking and secretion. A lot of critical proteins are involved in multiple steps of the process, such as Munc13-1 and Exocyst complex. Munc13-1 is an essential component controlling the vesicle priming event, whereas the Exocyst complex involves in regulating the process of vesicle terthering and docking. Recent works on these proteins properties and characters, as well as the 3D structures provided useful information for learning their molecular mechanism of function. A series of Munc13-1 truncations were rational designed based on homology and function, overexpressed in E. coli system, and purified to homogeneity with chromatography. In addition, the highα-helix contents of these constructs were measured by circular dichroism method, and their distinct oligomeric states, monodispersity and homogeneity properties were analyzed and compared by gel chromatography and analytical ultracentrifugation. Furthermore, a topology structure of long helical-bundle like of Munc13-1 and a model that Munc13 acts as a monomer in vivo were first raised by analysis of our data.
Through the trials of overexpression and purification of Sec10, one subunit of Exocyst complex, we concluded that Sec10 folds closely with its full-length amino acids because any truncation either in the amino (N)-terminal or in the carboxyl (C)-terminal induced error-folded structures, resulting in the formation of inclusion bodies. The highly polymerized Sec10 showed globular structure in solution by electron microscope.
GABAB Receptor belongs to class C family of G-protein coupled receptors, its function disorder results in lots of nervous diseases, such as Alzheimer, Parkinsonismus, et al. We used hypothermal expression and inclusion body denaturation/renaturation methods to express and purify enough yields of exocellular fragments of GABAB R for protein crystallization.
Glucose transporter 4 (GLUT4), as a hot spot protein in the research of type II diabetes, has a crucial role in whole-body glucose homeostasis. To data, there has little report on the method of large-scale extraction and purification of GLUT4. High yield of GLUT4 was extracted and purified from fat tissues by methods of homogenate, differential centrifugation and ammonium sulfate precipitation. The paper developed a method of effective purification of GLUT4 from fat tissue for further protein crystallization.
It is obvious that the bottleneck of membrane structural biology is the extraction, purification and crystallization of the membrane proteins. Recent works on the fields of trafficking and disease related membrane proteins in this paper shed new light on the further proteins crystallization.
GABAB受体属于G蛋白偶联受体中的C蛋白家族,与各类神经和精神错乱疾病有关。我们通过低温表达和包涵体变性复性实验得到了可用于结晶的具有药物结合靶点活性的胞外蛋白片断。葡萄糖转运蛋白GLUT4(Glucose Transporter 4)作为II型糖尿病领域研究中的关键蛋白,在维持体内糖平衡起到重要作用。目前世界上还未有关于体外组织大规模纯化GLUT4的相关报道。我们首次尝试选用猪脂肪作为原材料,采用组织匀浆,差速离心和硫酸铵沉淀方法初步分离纯化并获得了足够蛋白产量和纯度的GLUT4。
膜蛋白的纯化与结晶是当前结构生物学领域里的一大难题。当前蛋白表达提取,蛋白性质优化等研究为进一步的结晶学研究打下基础。
The process of vesicle translocation and fusion, as a ubiquitous and fundamental event in the cell growth and propagation, play a critical role in proteins trafficking and secretion. A lot of critical proteins are involved in multiple steps of the process, such as Munc13-1 and Exocyst complex. Munc13-1 is an essential component controlling the vesicle priming event, whereas the Exocyst complex involves in regulating the process of vesicle terthering and docking. Recent works on these proteins properties and characters, as well as the 3D structures provided useful information for learning their molecular mechanism of function. A series of Munc13-1 truncations were rational designed based on homology and function, overexpressed in E. coli system, and purified to homogeneity with chromatography. In addition, the highα-helix contents of these constructs were measured by circular dichroism method, and their distinct oligomeric states, monodispersity and homogeneity properties were analyzed and compared by gel chromatography and analytical ultracentrifugation. Furthermore, a topology structure of long helical-bundle like of Munc13-1 and a model that Munc13 acts as a monomer in vivo were first raised by analysis of our data.
Through the trials of overexpression and purification of Sec10, one subunit of Exocyst complex, we concluded that Sec10 folds closely with its full-length amino acids because any truncation either in the amino (N)-terminal or in the carboxyl (C)-terminal induced error-folded structures, resulting in the formation of inclusion bodies. The highly polymerized Sec10 showed globular structure in solution by electron microscope.
GABAB Receptor belongs to class C family of G-protein coupled receptors, its function disorder results in lots of nervous diseases, such as Alzheimer, Parkinsonismus, et al. We used hypothermal expression and inclusion body denaturation/renaturation methods to express and purify enough yields of exocellular fragments of GABAB R for protein crystallization.
Glucose transporter 4 (GLUT4), as a hot spot protein in the research of type II diabetes, has a crucial role in whole-body glucose homeostasis. To data, there has little report on the method of large-scale extraction and purification of GLUT4. High yield of GLUT4 was extracted and purified from fat tissues by methods of homogenate, differential centrifugation and ammonium sulfate precipitation. The paper developed a method of effective purification of GLUT4 from fat tissue for further protein crystallization.
It is obvious that the bottleneck of membrane structural biology is the extraction, purification and crystallization of the membrane proteins. Recent works on the fields of trafficking and disease related membrane proteins in this paper shed new light on the further proteins crystallization.
引文
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