CRYSTALLIZATION OF IL-3 AN ANTIBODY MEDIATED APPROACH

点击翻译按钮获取中文摘要

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.