| Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors belonging to the steroid/thyroid/retinoid receptor superfamily of ligand-activated transcription factors. Three PPARs subtypes have been identified thus far and are termed PPARα, PPARβ/δ, and PPARγ. The PPARγ receptor subtype, which is activated by polyunsaturated fatty acids and their metabolites, is predominantly expressed in adipose tissue and plays a pivotal role in lipid and glucose metabolism. Some potent synthetic PPARγ ligands have proven effective in the treatment of diabetes,and the use of such ligands has allowed researchers to unveil many potential roles for PPARγin pathological states including atherosclerosis, hypertension, inflammation, and cancer. These findings raise the exciting possibility that PPARγwill be an important target for the development of new drugs for the treatment of a wide variety of diseases.Random peptide libraries, which have allowed the identification of peptides that specifically bind to a variety of targets, are now widely used for mapping epitopes of antibodies, identifying peptide ligands, and developing vaccine candidates. To get bioactive peptides binding to PPARγ, the recombinant PPARγ-LBD protein was expressed and used as the target for biopanning from two random peptide libraries. Peptides with high affinity to PPARγ were screened and their biological function was analyzed. In summary, our work was conducted as follow:The fusion protein PPARγ-LBD was induced to express in E.coli. BL21(DE3). E.coli. cells were harvested after induction by 0.5mmol/L IPTG for 12 hours at 20℃ and then sonicated. The products were analyzed by SDS-PAGE. An obvious expression about 34kD can be detected and about half of the fusion protein was soluble. Purification was carried out with Ni2+-NTA agrose as there is a 6×His tag at the N-terminal of the recombinant protein. SDS-PAGE analysis showed that the target protein was highly purified and the purity was up to 90 percent.The purified protein coated on polystyrene plate was used as the target to carry out four rounds of bio-panning from phage-displayed random peptide libraries. Three positive phages were isolated from a 12-peptide library and five from a C7C-peptide library. Then the affinity of selected recombinant phage clones was detected by ELISA, and the DNA sequences of positive clones were determined, and finally amino acid sequences of 12-peptides and C7C-peptides were deduced. An 'LXXLL' motif was found in a 12-peptide with high affinity to PPARγand a consensus 'DXXRW' motif was included in C7C-peptides sequences.Rosiglitazone, as a potent PPARγ agonist, was used to carry out a competing ligand binding assay and did not have any effect on peptides' binding to PPARγ-LBD. It suggests that selected phage-displayed peptides have different binding sites on PPARγ-LBD from Rosiglitazone.Peptides binding to PPARγ were successfully screened and their biological function was analyzed. It may pave the way for further study of mechanism of PPARγ and novel therapeutic agents based on PPARγ.
|
PDF Full Text Request