Screening The Peptide Binding To Human Transferrin Receptor From Phage-display 12 Peptide Library

Human transferrin receptor (hTfR) is a II type transmembrane protein of which N terminal is in the cytoplasm and C terminal is out of the cytoplasm. It plays an important role in mediating the iron ion being uptaked by cells through endocytosis way. On the surfaces of tumor cells and the brain capillary endothelia cells, which consist of the brain blood barrier, the expressione levels of hTfR are high. And it has been proved that endocytosis is an efficient way to transport big molecules into cells. The two features mentioned above make hTfR an attractive target in drug delivery system and many researches have verified the feasibility of such strategy. Many therapeutic molecules such as anti-cancer drugs, proteins, peptides, DNA, PNA and so on can be delivered into cells with higher specificity through this method.hTfR has great potential in medical diagnosis and therapy, especially in treatment of cancer and central nervous system diseases.Phage-display technology is a powerful tool in finding the ligands for proteins, enzymes, antibodies and et al.In our study a phage-display 12 peptides library was screened with hTfR. After three cycles biopanning and phage ELISA, 8 positive phages were selected. DNA sequencing revealed that two consensus peptides sequences were included in the 8 positive phages, one is RXR and the other is RXXXR. The phage, which had highest A410nm absorbtion in phage ELISA was named as B18, and the peptide displayed was named as P18.The binding of B18 to hTfR is dose-dependent. Blast online analysis indicated that none homogeneouse sequence with PI 8 was found in proteins including Tf. Competition assay also demonstrated that the binding between B18 and hTfR could not be blocked by Tf which indicated that the binding site of B18 to hTfR is distinct from Tf, so the binding between Tf and hTfR will not be interfered with by B18. The assays of flow cytometry andcell immochemistry indicated that B18 has the ability to bind to SMMC-7721 on which the hTfR were highly expressed.When phage B18 and B26, a control phages were injected into the nude mice from tail vein, B18 recovered from tumor was as 5.23 times as B26, which indicated that B18 could home to tumor tissues specifically.In order to study whether the peptide still has the ability to direct other molecules to bind to hTfR when it was departed from phage, GST-P18 fusion gene was constructed and expressed in E.coli. The fusion protein was purified by affinity chromatography. The flow cytometry assay indicated that the fusion protein could bind to SMMC-7721 and when it was observed with a confocal laser-scanning microscopy, the imagines showed that the fusion protein was mainly binded on the cell membrane.