Isolation, Identification And Bioinformatics Analysis Of Intestinal Trefoil Factor Receptor

Background: Intestinal trefoil factor (ITF) is a small polypeptide, secreted abundantly onto the mucosal surface by the goblet cells of the gastrointestinal tract. ITF has one structurally characteristic'trefoil'domain which is defined as a sequence of 38-39 amino acid residues, in which six cysteines are disulphide-link in a 1-5, 2-4, and 3-6 configuration. The amino acid sequence, together with the disulphide bonds, forms a characteristic three-leaved structure which has given the peptide its name. ITF appears to play an important role in the protection, repair and healing of the gastrointestinal mucosa. It has intrinsic activity in regulating intestine epithelium proliferation and migration. Studies showed that cells migrated more rapidly from the crypts to the villus tips in wild-type animals compared with ITF-null mice. Studies have also shown that ITF increased the resistance of colonic epithelium to apoptosis induced by etoposide. Taken together, these observations suggest that ITF is an important cytoprotection and anti-apoptotic peptide in the GI tract, and several signal transduction pathways have been associated with ITF biological actions. However, it is not clear the detailed signaling mechanism, especially post-receptor signaling mechanism. Some reports suggested that ITF actions via its autoreceptor. The evidence of putative candidate ITF receptor proteins has been reported recently and our primary experiments indicated that ITF has its receptor (ITFR). Up to now,ITFR has not been finally identified yet.Objective: To isolate ITFR from preparations of cell membrane by pull-down assay and identify ITFR with mass spectrographic analysis, co-immunoprecipitation, yeast two-hybrid and bioinformatics analysis.Methods:Expression and purification of the rhITF fusion protein in Pichia pastoris : Successfully transformed Pichia pastoris strain X-33 which contained ITF gene encoding mature peptide were screened with Zeocin. Subsequently, ITF was constitutively expressed and purified by ammonium sulfate precipitation, Ni+–NTA affinity chromatography and ultrafiltration, determined by SDS-PAGE and Western blot analysis.Expression and purification of the rhITF fusion protein in E.coli: hITF gene was obtained by PCR, and then inserted into the MCS of the expression vector pGEX-4T-1 to construct the recombinant vector. After identification by double enzyme digestion and gene sequencing, the recombinant vector was transformed into E.coli BL21(DE3),and rhITF fusion protein was expressed by IPTG induction. The rhITF fusion protein was purified by GST agarose affinity chromatography, determined by SDS-PAGE,Western blot and mass spectrographic analysis.Isolation of ITFR: As a bait,the purified recombinant ITF contained GST tag or His tag was binded to affinity column, then incubated with preparation of cell membrane, ITFR was eluted from affinity columned and isolation by SDS-polyacrylamide gel electrophoresis and indentified by mass spectrographic analysis. By using bioinformatics software, analyse the data from mass spectrographic analysis for physical and chemical properties, transmembrane domain, grand average of hydrophobicity, instability index, and search literature to find experimental evidences for candidate proteins of ITFR.Yeast two-hybrid screening interacting protein: Construction of bait plasmids and intestinal epithelial cell library, co-transformed into yeast Mav203, then the transformed Mav203 cells were grown in SC-Trp-Leu-His+3AT mediums for screening positive transformants. After characterization, the prey vectors were separated, sequenced and blasted in NCBI.Results:1. ITF was successfully expressed in Pichia pastoris, Tricine SDS-PAGE and Western blot analysis showed that ITF had much good antigenicity and specificity. The purity after purification was above 95%, the yield of ITF about 50mg/L.2. As a result of PCR, the 215bp DNA fragment was obtained and cloned into vetor pGEX-4T-1, the recombinant vetor was named pGEX-4T-1-hITF, the size of the amplification fragment was consistent with the theoretical value (GenBank Accession No. NM₀03226). The recombinant vector was successfully constructed and the rhITF fusion protein was expressed at a concentration of 120mg/L, the purity was above 95%.Western blot analysis and mass spectrographic analysis showed that the rhITF fusion protein had considerable antigenicity and specificity.3. A 35kD protein was isolated by pull-down assay,by using monoclonal antibody of ITF,the same molecular mass protein was also obtained from co-immunoprecipitation experiments. The 35kD protein was analyzed by mass spectrographic and 95 proteins were obtained as candidate proteins. Bioinformatics analysis data show three proteins PDZD2, Wnt8b and ITIH4 may be ITF receptor.4. Bait Vector and intestinal epithelial cell library were successfully constructed. After screening, four proteins PDZD2,TIMP1,CD44 antigen and VPS4-1 were obtained by yeast two-hybrid screening interacting protein.Conclusion:1. The rhITF fusion protein was successfully produced by genetic engineering.2. A protein that molecular mass about 35kD was isolated by pull-down assay. PDZD2, Wnt8b and ITIH4 as candidate proteins of ITF receptor were analyzed and identified by mass spectrographic and bioinformatics analysis.3. TIMP1,CD44 antigen,PDZD2 and VPS4-1 as ITF interacting protein were screened by yeast two-hybrid.PDZD2, the most likely ITFR ,was eventually identified.