Screening Human Transferrin Specific Binding Peptides By Phage Display Technology

The transferrin (Tf) is an essential serum glycoprotein mostly known for its iron transport capacity. The transferrin receptor (TfR) is not only involved in the cellular uptake of iron but also in the regulation of cell growth. The elevated expression levels of TfR in malignancies make it an effective tumor biomarker and an excellent cancer therapy target. For targeted tumour therapy, transferrin has been used as a drug carrier targeting the TfR by receptor-mediated endocytosis in order to delivery therapeutic agents such as chemotherapy drugs, protein toxins or therapeutic gene into tumor cells in order to increase the specificity of the drug binding capacity and improve the therapeutic effect.We use phage display technology to screen transferrin binding peptides (TfB) from a phage fused random heptapeptide library for the transferrin-drug conjugates prepared by coupling through noncovalent bond. TfB will be fused with protein drug for binding to transferrin as tumor-targeting therapy possibly.Bio-banning is carried out by incubating a library of phage-displayed peptides with a plate coated with the target (Tf), washing away the unbound phage, and eluting the specifically-bound phage with Glycine-HCl (pH2.2). The eluted phage is then amplified and taken through additional binding/amplification cycles to enrich the pool in favor of binding sequences.Individual clones are isolated, indentified and sequenced by DNA. We developed the selection criteria of recovery yield and specificity ratio in the panning process. Recovery yield is the ratio of the number of eluted phage and input phage, specificity ratio is the number of eluted phage capable binding to the target molecule coated plate to the blank control plate. When two parameters increased in each round, positive recombinant phage clones should be enriched. In addition, we developed a direct measurement via phage titres of the relative affinity constants. A double reciprocal plot is generated by simply using the titre of input phage and eluted phage to calculate Kd.After three rounds of biopanning, positive recombinant phage clones were enriched. The relative affinity constants of the four positive TfB clones are at the micromolar level, which were assayed with the measurement based on phage titres. DNA sequencing found the different sequences of these four clones.Therefore TfB will be linked Tf through noncovalent bond. for targeted drug systems and be fused with protein drug for extending protein half life.