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 (~50aa) -Linker(~120aa) -EFD2(~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.
Thesaurus Terms: colitis, drug design /synthesis /production, gastrointestinal agent, mucin, protein engineering cell migration, chimeric protein, disease /disorder model, wound healing biotechnology, laboratory mouse
