Production Of Human Intestinal Trefoil Factor In Escherichia Coli And Pichia Pastoris And Its Effect On The Restitution Of Gastrointestinal Mucosal Arrier

Background: Intestinal trefoil factor (ITF), a member of the trefoil factor family (TFFs), was discovered and named by Suemori in 1991. ITF is composed of one structurally characteristic trefoil domain. A trefoil domain is defined as a sequence of 38 or 39 amino acid residues in which 6 cysteine residues are linked in the configuration 1-5, 2-4, 3-6, thus forming a characteristic three-leaved structure. ITF is essential for protecting the epithelial layer of the gastrointestinal tract from damage and repairing epithelium after injury. ITF is attracting the researchers' attention for its potential pharmacological value. Three methods are generally available for producing ITF: peptides synthesis, preparation from natural sources and recombinant technology. Due to the fact of expensive cost and low yield, peptides synthesis and preparation from natural sources could not be used to produce ITF on a large scale. And it was reported that an Escherichia coli expression system had been used for the production of ITF and the expression level was 3-4mg/L. Also, ITF was produced by a yeast expression system using saccharomyces cerevisiae with a yield of 100mg/L. As a yeast, Pichia pastoris shares the advantages of molecular and genetic manipulations with Saccharomyces cerevisiae , and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia pastoris very useful as a protein expression system. So far, P.pastoris has developed into a highly successful system for the production of a variety of heterologous proteins, such as tetanus toxin fragment C, enterokinase, human tumor necrosis factor and so on.Objective: To select an optimal expression system, obtain rhITF with high yield and high purity; to assay the stability of rhITF in an in vitro simulated gastrointestinal environment; and to explore its biological activity and restorative effect on gastrointestinal mucosal barrier.Methods:1. Expression and purification of rhITF in E.coli. hITF gene encoding mature peptide...