Quantitative Screening Human Serum Albumin Binding Peptides Using Phage Display Random Peptide Library

Human Serum Albumin (HSA) contributes about50%-60%of total protein in human plasma, and has a half-life of19days. It maintains blood osmotic pressure, transports nutrients and protects other important biological substances in human body. Moreover, the nanometer sized drug with HSA as carrier is gradually springing up. It can improve the stability of drugs and make drugs release slow, so that the action time will be prolonged, and the targeting transport will be accomplished.Not only HSA fusion protein but also HSA binding protein has become general strategy for improving the pharmacokinetics of proteins. The keys of the way to prolong half-lives of protein drugs are high affinity and specificity of HSA binding peptides. In this thesis, with HSA as the target molecules, Glycine-HCl (pH2.2) as elution solution to biopan the Ph.D.-7phage random peptides library, and with recovery yield and specificity ratio as the way to optimize the panning process quantitatively. Recovery is the ratio of the number of eluted phage and input phage; selectivity ratio (P/B) is the number of phages binding in the HSA-coated plate and the phages binding in the blank plate. Through4rounds coating, binding, elution and amplification, the yield and selectivity have a certain increase, indicating specific binding obtained with the target molecules of exogenous proteins or peptides. Furthermore, it can also preliminary screen the HSA binging peptide with selection ratio.Monoclonal phage were picked for specific identification and detect the relative affinity of the picked phage positive clones using the phage titer successfully. Finally, we got four specific peptide-binding sequences and found out that there is no homologous sequence among them, it reveals that they maybe adherence with different positions of HSA. In present paper, the affinity determinated by phage tier measuring technique is0.73μM,0.42μM、0.81μM�'�10.15μM.The use of phage display is highlighted for exploring protein-protein interactions in functional genomics in this article. Although antibody library display on phage has been very successful, phage display with genomic cDNA libraries is rare and inefficient compared with yeast two hybrid method. This article summarizes strategies including C-terminal display and ORF cDNA libraries to enables phage display identification of real endogenous proteins with efficiency, sensitivity and accuracy. We compare the strengths and weakness of phage display and yeast two hybrid methods. The application of phage display for screening functional genes and mapping protein-protein interactions in host-pathogen, immunogenic proteins of tumour antigens and allergens, ligands involved in signal transduction, small-molecule binding proteins and enzymes are also reviewed.