Novel Proteins for Treatment of Bowel Diseases

描述（由申请人提供）：本i期申请的目标是优化治疗肠道疾病（IBD）的蛋白质的活性并使其尺寸最小化，以便能够生产人体试验所需的大量蛋白质。目前，没有安全有效的治疗IBD的方法。明尼苏达大学的Samuel Ho教授和他的同事们已经制造出了一种天然存在的小鼠肠道粘蛋白的一部分，这种粘蛋白已知对IBD有保护作用，在小鼠结肠炎模型中是有效的。这种GST标记的蛋白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(279aa)-EFD1（约50aa） -Linker（约120aa） -EFD2（约60aa）。尽管这些EFD单位与EGF相似，但黏液衍生蛋白通过一种不涉及EGF受体激活的机制促进伤口愈合，因此降低了其潜在的致癌性。临床试验所需的大量生产这种大蛋白存在问题，本提案的具体目标是通过探索从人粘蛋白中提取不同EFD单元的构建物、截断连接段和去除GST标签来优化当前的候选蛋白。三叶草因子是人体肠道中自然产生的EFD单位，可促进伤口愈合。这些变体，连同人黏液蛋白MUC3和MUC17的EFD单元，将通过盒式诱变纳入黏液蛋白结构格式。为了达到MW < 15,000的目标，连接子段将被截断到符合生物活性保留的最短长度。Proteos将通过细菌表达产生缺乏纯化标签的蛋白质，如果形成包涵体，Proteos科学家已经成功地将这些蛋白质重新折叠。构建体将在细胞迁移和增殖试验中进行检测，并在明尼苏达大学Ho实验室应用于第一代小鼠粘蛋白早期研究的溃疡性结肠炎小鼠模型中进行检测。

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.