CRYSTALLIZATION OF IL-3 AN ANTIBODY MEDIATED APPROACH

描述：(改编自申请人的摘要)白介素3将 使用一种新的方法结晶，这可能提供一种通用的方法 产生重组蛋白质的晶体。人类IL-3将被生产出来 在大肠杆菌中作为含有短亲水性标志物的融合蛋白 多肽序列(Flag多肽)，融合到其氨基末端。这八个 氨基酸标志序列Asp-Tyr-Lys-Asp-Lys已显示 使含有FLAG的融合蛋白纯化到均一状态 抗Flag单抗亲和层析柱的研制 极端温和的条件(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 高纯度和活性的融合蛋白产品，两者都是 对结晶至关重要。一旦旗帜IL-3融合 蛋白质已经提纯，将进行结晶试验，两者 关于Flag-IL-3产物及其与Fab片段的络合物 相同的抗FLAG单抗。因为Fab片段和Fab/抗原复合体 都相对容易结晶，很可能会形成络合物 会改变Flag-IL-3分子的结晶性。成功 FLAG IL-3的结晶将服务于(L)提供 解决IL-3三维结构的起始材料 (2)检测FLAG抗体介导的结晶的实用性 概念。得到的晶体将被用来确定三个 第二期研究中IL-3的空间结构。这个结构，反过来， 可用于进行合理的药物设计程序，寻找新的和 改进的IL-3的模拟物或拮抗剂，将具有临床和 商业价值在治疗相关疾病中的作用 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.