Study On Screening Human Serum Albumin Binding Peptides By Phage Display Technology

In this thesis, phage display technology (PDT) was used to screen human serum albumin (HSA) peptide, which will be inserted into the protein drugs to prolong their half-lives in the serum.PDT is a powerful selection technique for screening polypeptides with novel proterties. A gene of interest is fused to that of a phage coat protein gene, resulting in phage particles that display the encoded protein and contain its gene, providing a direct link between phenotype and genotype. This allows phage libraries to be subjected to a selection step and recovered cloned to be identified by sequencing.Using Ph.D.-7phage display random peptide library, we identified a series of peptides that specially bind to HSA. In the experiment, panning was carried out by incubating a library of phage displayed peptides with a plate coated with the target of HSA, washing away unbound phage, and eluting the specifically bound phage. The eluted phage is then amplified and taked through additional binding/amplification cycles to enrich the pool in favor of binding sequences. After2-3rounds, individual clone was characterized by DNA sequencing. The recovery yield and specificity ratio have been defined as the selection criteria in the panning process. The recovery yield is the ratio of the number of eluted phage and input phage. Specificity ratio is the number of phages binding in the target plate to the phages binding in the blank plate. Both coefficients of the criterias should increase to obtain positive colnes. In order to achive high performance of panning process, two different elution, using Glycine-Hcl (pH2.2) as acid elution method and HSA as competive elution method, were carried out and compared. Finally, more than five postive clones with high specificify raio have been identified and characterize by DNA sequencing.HSA is an ideal long-acting carrier protein in drug design for prolonging the half-lives of protein drugs. Not only HSA fusion protein but also HSA binding protein has become general strategy for improving the pharmacokinetics of proteins. Comparision between the two methods were also discussed. HSA fusion protein has longer half-life, but its biological activity is siginificantly decreased. However, HSA binding protein keep its biological activity basicly unchanged while prolong its half-life.