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; 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，充当IgG的高亲和力受体。它参与了吞噬作用的激活，IgG揭露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的结构及其与免疫球蛋白的复合物。为了表达和纯化重组CD64蛋白，我们同时构建了细菌和基于CHO细胞的表达系统。基因扩增后，CHO细胞表达的产量保持在50 ug/升的细胞培养水平。另一方面，细菌系统以非活性包容体形式产生蛋白质。随后的重新折叠实验导致的蛋白质少于毫克的结晶。目前，我们正在尝试使用杆状病毒表达系统表达重组CD64。 B细胞通过形成由免疫球蛋白（Ig）分子和Ig Alpha（IgA）和Igβ（IGB）组成的抗原交联B细胞受体（BCR）的信号传导。为了阐明IGA与IGB之间以及异二聚体与BCR之间的相互作用，我们表达了E. Coli中IgA和IGB的细胞外域，作为包容体。每种蛋白质均编码在PET-30载体中，并包括C末端6 his标签以进行纯化，并获得了IgA和IGB包含体的高产量（培养物的15-50 mg/L）。试图单独或共同重新分配IGA和IGB。通过Ni-NTA和凝胶过滤色谱法纯化后，单独的IgA重折叠从50 mg包含物体中产生约2 mg的纯蛋白。通过质谱和SDS-PAGE分析确定，纯化的蛋白作为单体和同型IgA的混合物存在。 Iga-iga二聚体很可能是非共价的。纯化的IgA用于抗体生产，还进行了结晶试验。但是，样品似乎是不稳定的，沉淀在4？c以上，并且没有结晶筛网获得命中。单独的IGB重折叠无法产生大量的蛋白质降解。通过标准稀释方法将IGA和IGB共同折叠的初步尝试也因IGB降解而困扰。然而，通过在重折叠过程中将IgA和IGB包含物体隔离在一起，我们通过Ni-NTA亲和色谱和阴离子交换单体和二聚体IgA的混合物以及IgA-IgA-IgB异二聚体纯化。我们还使用细菌表达/再折叠系统来表达鼠Iga-igb。