Novel Proteins for Treatment of Bowel Diseases

描述（由申请人提供）：本1期提案的目标是优化活性并最小化用于肠道疾病（IBD）的蛋白质治疗剂的大小，以便能够生产人体试验所需的蛋白质。目前，IBD还没有安全有效的治疗方法。明尼苏达大学的Samuel Ho教授及其同事已经产生了一部分天然存在的鼠肠道粘蛋白，已知其对IBD具有保护作用，在结肠炎小鼠模型中有效。这种GST标记的蛋白质含有两个类似于表皮生长因子（EGF）的小结构域，我们称之为EFD单元，由肽段连接，即：GST（279 aa）-EFD 1（约50 aa）-接头（约120 aa）-EFD 2（约60 aa）。尽管这些EFD单位与EGF相似，但粘蛋白衍生蛋白通过不涉及EGF受体活化的机制促进伤口愈合，因此降低其致癌性的可能性。临床试验所需数量的这种大蛋白的生产存在问题，该提案的具体目的是通过探索具有来自人粘蛋白的不同EFD单元的构建体、接头片段的截短和GST标签的去除来优化当前候选蛋白。三叶因子是天然存在的EFD单位，在人体肠道中产生，可促进伤口愈合。这些变体与人粘蛋白MUC 3和MUC 17的EFD单位一起，将通过盒式诱变掺入粘蛋白结构形式中。为了达到MW <15，000的目的，将接头区段截短至与保留生物活性一致的最短长度。缺乏纯化标签的蛋白质将在Proteos通过细菌表达产生，如果形成包涵体，Proteos的科学家已经成功地将这些蛋白质重折叠。构建体将在细胞迁移和增殖测定中进行测定，并在明尼苏达大学Ho实验室在第一代鼠粘蛋白的早期研究中应用的溃疡性结肠炎小鼠模型中进行测定。

DESCRIPTION (provided by applicant): The goal of this Phase 1 proposal is to optimize the activity and minimize the size of a protein therapeutic for intestinal bowel diseases (IBD), in order to enable production of the protein in quantities needed for trials in humans. Currently, there is no safe and effective treatment for IBD. Professor Samuel Ho and colleagues at the University of Minnesota have produced a portion of a naturally occurring murine intestinal mucin known to be protective against IBD that is effective in a mouse model of colitis. This GST-tagged protein contains two small domains that are similar to epidermal growth factor (EGF) and that we refer to as EFD units, linked by a peptide segment i.e.: GST(279aa)-EFD1 (approximately 50aa) -Linker(approximately 120aa) -EFD2(approximately 60aa). Despite the similarity of these EFD units to EGF, the mucin-derived protein promotes wound healing by a mechanism that does not involve activation of the EGF receptor, therefore lowering its potential for carcinogenicity. There are problems with production of this large protein in quantities needed for clinical trials, and the specific aims of this proposal are to optimize the current candidate protein by exploration of constructs with different EFD units from human mucins, truncation of the linker segment, and removal of the GST tag. Trefoil factors are naturally occurring EFD units that are produced in the human intestine and that promote wound healing. These variants, together with the EFD units of human mucins MUC3 and MUC17, will be incorporated by cassette mutagenesis into the mucin structural format. With the objective of achieving a MW < 15,000, the linker segment will be truncated to the shortest length consistent with reservation of biological activity. Proteins lacking purification tags will be produced at Proteos by bacterial expression and if inclusion bodies are formed, Proteos scientists have been successful in refolding such proteins. Constructs will be assayed in cell-migration and proliferation assays, and in the mouse model of ulcerative colitis applied in the Ho laboratory at the University of Minnesota in earlier studies of the first generation murine mucin protein.