CRYSTALLIZATION OF IL-3 AN ANTIBODY MEDIATED APPROACH

描述：（改编自申请人的摘要）白细胞介素3将是 结晶使用新的方法，可以提供一个通用的方法， 产生重组蛋白的晶体。 人IL-3将在 在大肠大肠杆菌作为含有短亲水标记的融合蛋白 肽序列（Flag肽），与其氨基末端融合。 这个八 已显示氨基酸Flag序列Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys 为了使含Flag的融合蛋白在细胞上纯化至均一， 将抗Flag单克隆抗体（MAb）的亲和柱在 极其温和的条件（磷酸盐缓冲盐水，pH 7.2，+或- 钙）。 这种独特而温和的一步纯化程序产生Flag 高纯度和高活性的融合蛋白产物， 对结晶至关重要。 一旦Flag IL-3融合 蛋白质已经纯化，将进行结晶试验， 游离Flag-IL-3产物以及其与Fab片段的复合物 相同的抗Flag单克隆抗体 因为Fab片段和Fab/抗原复合物 相对容易结晶，很可能形成复合物 将改变Flag-IL-3分子的结晶性。 成功 Flag IL-3的结晶将服务于以下双重目的： 用于解析IL-3的三维结构的起始材料 以及（2）测试Flag抗体介导的结晶的效用 概念. 获得的晶体将用于确定三个 II期研究中IL-3的三维结构。 这种结构，反过来， 可用于进行合理的药物设计程序， IL-3的改进的模拟物或拮抗剂， 在治疗相关疾病方面的商业价值作用 IL-3在早期造血和免疫中的作用。

DESCRIPTION: (Adapted from the applicant's abstract) Interleukin 3 will be crystallized using a new approach that may provide a general method for generating crystals of recombinant proteins. Human IL-3 will be produced in E. coli as a fusion protein containing a short hydrophilic marker peptide sequence (Flag peptide), fused to its amino terminus. This eight amino acid Flag sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, has been shown to allow Flag-containing fusion proteins to be purified to homogeneity on an affinity column of an anti-Flag monoclonal antibody (MAb) under extremely mild conditions (phosphate buffered saline, pH 7.2, + or - calcium). This unique and mild one-step purification procedure yields Flag fusion protein products of high purity and activity, both of which are critically important for crystallization. Once the Flag IL-3 fusion protein has been purified, crystallization trials will be carried out, both on the free Flag-IL-3 product and also on its complex with the Fab fragment of the same anti-Flag MAb. Because Fab fragments and Fab/antigen complexes are relatively easy to crystallize, it is likely that forming the complex will alter the crystallizability of the Flag-IL-3 molecule. Successful crystallization of Flag IL-3 will serve the dual purposes of (l) providing the starting material for solving the three dimensional structure of IL-3 and (2) testing the utility of the Flag antibody mediated crystallization concept. The crystals obtained will be used to determine a three dimensional structure of IL-3 in Phase II study. This structure, in turn, can be used to carry out rational drug design procedures to find new and improved mimics or antagonists for IL-3, which will be of clinical and commercial value in the treatment of diseases that are related to the role of IL-3 in early hematopoiesis and immunity.