Novel Proteins for Treatment of Bowel Diseases

描述(由申请人提供)：这一阶段提案的目标是优化活性并将治疗肠道疾病(IBD)的蛋白质的大小降至最低，以便能够生产出人体试验所需的蛋白质。目前，还没有安全有效的治疗IBD的方法。明尼苏达大学的Samuel Ho教授和他的同事已经生产出一种自然产生的小鼠肠道粘蛋白的一部分，这种粘蛋白对IBD有保护作用，对结肠炎的小鼠模型有效。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单位，由一个肽段连接，即：GST(279aa)-EFD1(约50aa)-Linker(约120aa)-EFD2(约60aa)。尽管这些EFD单位与EGF相似，但粘蛋白衍生的蛋白通过一种不涉及EGF受体激活的机制促进伤口愈合，从而降低其致癌潜力。临床试验所需的大量生产这种大蛋白存在问题，这一提议的具体目的是通过探索来自人粘蛋白的不同EFD单位的结构、截断连接片段和去除GST标签来优化当前候选蛋白。三叶因子是自然产生的EFD单位，在人体肠道中产生，促进伤口愈合。这些变异体与人类粘蛋白MUC3和MUC17的EFD单元一起，将通过盒式突变被整合到粘蛋白结构形式中。为了达到15,000 MW&lt；的目标，连接体片段将被截断到与生物活性保留一致的最短长度。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对这些蛋白质进行了复性。构建物将在细胞迁移和增殖分析中进行分析，并在明尼苏达大学HO实验室应用于第一代小鼠粘蛋白的早期研究中用于溃疡性结肠炎的小鼠模型中。

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.