Expression of Human Intestinal Trefoil Factor in Escherichia coli.

BROWN, L.; HEBERT, E.M.; RAYA, R.R.

Congreso; VII Congreso Argentino de Microbiología General “SAMIGE del Bicentenario.; 2011

Lactic acid bacteria (LAB) are used as a delivery system for a range of molecules that have different applications, including therapies for allergic and gastrointestinal diseases. Intestinal trefoil factor (ITF), a small, compact protease-resistant peptide, is abundantly expressed in goblet cells of large and small intestine. Several biological activities of ITF have been identified, including promotion of wound healing, stimulation of epithelial cell migration, and protection of intestinal epithelial barrier. The therapeutic use of recombinant ITF has been suggested for the treatment of patients with chronic inflammatory bowel diseases, peptic ulcer, necrotizing enterocolitis and gastrointestinal surgery. However, one of the factors that limit its use is the high cost associated to their production and purification. Furthermore, when the ITF is administered orally, it adheres to the mucus of the small intestine being absorbed in the cecum. On the contrary, intragastric administration of ITF by the action of recombinant LAB would lead to the release of active peptides in the mucosa of colon. The aim of this study was the expression of human ITF in <i>Escherichia coli </i> forits subsequent expressioninLAB for the preventionandtreatmentof intestine damage. HumanITF gene encoding mature peptide was obtained by RT-PCR using the plasmid pSport (Clontech) as template. The amplified fragment (approximately 200 bp) was cloned into pBlueScript SKII (Stratagene) resulting the plasmid pSal. By using specific primers with cut sites for <i>Sal</i>I and <i>Nco</i>I the ITF was amplified and inserted into the expression vector pET28b to construct the recombinant pET28b-ITF. After confirmation by gene sequencing, pET28b-ITF was transformed into <i>E. coli</i> BL21(DE3), and the fusion protein was expressed by isopropyl-b-d-thiogalactopyranoside induction in shake flask and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The subsequent expression of ITF fusion in LAB is ongoing, constituting the recombinant LAB strain a promising tool for the treatment of intestinal damage.