Binding Kinetics of LRP Protein with Receptor Related Protein

LRP 蛋白与受体相关蛋白的结合动力学

• 批准号: 7966043
• 负责人: Sandra Smith-Gill
• 金额: $ 17.43万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7966043
• 关键词: Address; Binding; Binding Sites; Biological Assay; Cell physiology; Chimeric Proteins; Cloning Vectors; Collaborations; Complement; Complex; Cysteine; Escherichia coli; Family; Fluorescence; In Vitro; Kinetics; Label; Laboratories; Ligand Binding; Ligands; Lipoprotein Receptor; Molecular; Molecular Target; Oncogenic; Pathway interactions; Protein Binding; Proteins; Resolution; Role; Signal Pathway; Structure; Tumor Suppressor Proteins; Ubiquitin; Work; cancer therapy; chaperonin; extracellular; gene cloning; in vivo; insight; member; protein complex; receptor

This is a collaboration with Y-X Wang (SBL) to optimize expression of continuous repeat unit CR567 of Lipoprotein Receptor Related Protein (LRP)in order to study its binding kinetics with RAP (Receptor Related Protein)and to determine the structure of the C567-RAP complex. LRP is a transmembrane receptor protein that binds with more than 30 ligands in vivo, making it an important receptor in many cellular processes. LRP has many structural domains; domain II contains several continuous repeat (CR) units including CR567. Previous studies have shown that the CR567 units are important for RAP binding, although the role of the specific CR units in RAP binding is still poorly understood. The signaling pathway of LRP is tightly controlled by a number of extracellular and intracellular regulating proteins, including chaperonins and members of the Dickkopf (DKK) family. These proteins down-regulate Wnt-B-catenin pathway as tumor suppressors. LRP6 has been shown to be an oncogenic receptor. This pathway has high potential for molecular targeting. The ligand binding sites of CR567 are cysteine-rich complement type repeats, making expression and purification with proper folding difficult, a major hurdle to biophysical and structural studies. Other laboratories have expressed CR56 as a ubiquitin fusion protein, which is the approach we are using to express CR567. We have cloned the gene encoding CR567 into pET SUMO cloning vector also containing throredoxin, and expressed protein using the origami E. coli strain. The resultant protein can be separated from aggregates, is clear when labeled with N15, and gives a better resolution NMR spectrum than has been obtained previously. We are working on binding assays using SPR,intrinsic fluorescence, and ITC. This work will be continued in Dr. Wangs laboratory after closure of my section at the end of FY09.

这是与Y-X Wang（SBL）的合作，以优化脂蛋白受体相关蛋白（LRP）连续重复重复单位CR567的表达，以研究其与RAP（受体相关蛋白）的结合动力学，并确定C567-RAP复合物的结构。 LRP是一种跨膜受体蛋白，在体内与30多个配体结合，使其成为许多细胞过程中的重要受体。 LRP有许多结构域。域II包含几个连续重复（CR）单元，包括CR567。先前的研究表明，CR567单位对于RAP结合很重要，尽管特定CR单元在RAP结合中的作用仍然很少了解。 LRP的信号通路受许多细胞外和细胞内调节蛋白的紧密控制，包括伴侣蛋白和Dickkopf（DKK）家族的成员。这些蛋白质将Wnt-B-catenin途径下调为肿瘤抑制剂。 LRP6已被证明是一种致癌受体。该途径对于分子靶向具有很高的潜力。 CR567的配体结合位点是富含半胱氨酸的补体类型的重复，使表达和纯化变得困难，这是生物物理和结构研究的主要障碍。其他实验室已将CR56表示为泛素融合蛋白，这是我们用来表达CR567的方法。我们将编码CR567的基因克隆到含有throedoxin的PET Sumo克隆载体中，并使用折纸大肠杆菌菌株表达蛋白质。当用N15标记时，可以将所得蛋白与聚集体分离，并且比以前获得的分辨率NMR光谱更好。我们正在使用SPR，固有荧光和ITC进行结合测定。在2009财年结束时关闭我的部门后，王博士实验室将继续这项工作。