Structural Determination-Fcreceptor CD64/B cell receptor

结构测定-Fc受体 CD64/B细胞受体

• 批准号: 7196656
• 负责人: PETER D. SUN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/7196656
• 关键词: B cell receptor; CHO cells; Escherichia coli; X ray crystallography; antibody receptor; gel filtration chromatography; immunoglobulin A; immunoglobulin G; immunologic receptors; protein folding; protein protein interaction; protein purification; receptor expression; recombinant proteins; structural biology; surface plasmon resonance; transfection /expression vector

CD64, also known as the type I Fc-gamma receptor, functions as a high affinity receptor for IgG. It is involved in the activation of phagocytosis, endocytosis of IgG-opsonized particles, and activation of the complement system. Our goal is to determine the structure of CD64 and its complex with immunoglobulins. To express and purify the recombinant CD64 protein, we have constructed both bacteria and a CHO cell based expression system. The yield of the CHO cell expression remained at a level of 50 ug/liter of cell culture after gene amplification. The bacteria system, on the other hand, yielded the protein in an inactive inclusion body form. Subsequent refolding experiments resulted in less than a mg of the protein for crystallization. Currently, we are attempting to express the recombinant CD64 using a baculovirus expression system. B cells initiate their signaling through the formation of antigen cross-linked B cell receptor (BCR) which consists of an immunoglobulin (Ig) molecule and Ig alpha (Iga) and Ig beta (Igb). In efforts to elucidate the interaction between Iga and Igb and between the heterodimer and the BCR, we have expressed the extracellular domains of Iga and Igb in E. coli as inclusion bodies. Each protein was encoded in a pET-30 vector and included a C-terminal 6-His tag for purification, and high yields of both Iga and Igb inclusion bodies were obtained (15-50 mg/L of culture). Attempts were made to refold Iga and Igb either individually or in concert. Refolding of Iga alone yielded approximately 2 mg of pure protein from 50 mg of inclusion bodies following purification by Ni-NTA and gel filtration chromatography. The purified protein was present as a mixture of monomeric and homodimeric Iga as determined by mass spectrometry and SDS-PAGE analysis; the Iga-Iga dimer was most likely noncovalent. Purified Iga was used for antibody production and was also subjected to crystallization trials. However, the sample appeared to be unstable, precipitating above 4?C, and no hits were obtained with crystallization screens. Refolding of Igb alone failed to yield significant quantities of protein due to degradation. Initial attempts to co-refold Iga and Igb together by standard dilution refolding methods were also plagued by degradation of Igb. However, by sequestering Iga and Igb inclusion bodies together in a smaller volume during refolding, we purified by Ni-NTA affinity chromatography and anion exchange a mixture of monomeric and dimeric Iga, and the Iga-Igb heterodimer. We are also expressing the murine Iga-Igb using a bacteria expression/refolding system.

CD64，也称为 I 型 Fc-gamma 受体，是 IgG 的高亲和力受体。它参与吞噬作用的激活、IgG 调理颗粒的内吞作用以及补体系统的激活。我们的目标是确定 CD64 及其与免疫球蛋白复合物的结构。为了表达和纯化重组 CD64 蛋白，我们构建了细菌和基于 CHO 细胞的表达系统。基因扩增后，CHO细胞表达的产量保持在50ug/升细胞培养物的水平。另一方面，细菌系统产生无活性包涵体形式的蛋白质。随后的重折叠实验产生了不到一毫克的蛋白质结晶。目前，我们正在尝试使用杆状病毒表达系统来表达重组CD64。 B 细胞通过形成抗原交联 B 细胞受体 (BCR) 来启动信号传导，该受体由免疫球蛋白 (Ig) 分子以及 Ig α (Iga) 和 Ig β (Igb) 组成。为了阐明 Iga 和 Igb 之间以及异二聚体和 BCR 之间的相互作用，我们在大肠杆菌中将 Iga 和 Igb 的胞外结构域表达为包涵体。每种蛋白均在 pET-30 载体中编码，并包含用于纯化的 C 端 6-His 标签，并获得高产率的 Iga 和 Igb 包涵体（培养物 15-50 mg/L）。人们尝试单独或共同重新折叠 Iga 和 Igb。经过 Ni-NTA 和凝胶过滤层析纯化后，仅 Iga 的重折叠从 50 mg 包涵体中产生约 2 mg 纯蛋白。通过质谱和 SDS-PAGE 分析确定，纯化的蛋白质以单体和同二聚 Iga 的混合物形式存在； Iga-Iga 二聚体很可能是非共价的。纯化的 Iga 用于抗体生产，并进行结晶试验。然而，样品似乎不稳定，在 4°C 以上会沉淀，并且通过结晶筛选未获得命中。由于降解，仅 Igb 的重折叠无法产生大量蛋白质。最初通过标准稀释重折叠方法将 Iga 和 Igb 共重折叠在一起的尝试也受到 Igb 降解的困扰。然而，通过在重折叠过程中将 Iga 和 Igb 包涵体隔离在较小的体积中，我们通过 Ni-NTA 亲和层析和阴离子交换纯化了单体和二聚体 Iga 以及 Iga-Igb 异二聚体的混合物。我们还使用细菌表达/重折叠系统表达鼠 Iga-Igb。