第七章-2 展示系统.pptVIP

Chapter 7 Techniques for study protein-protein interaction --Display system Phage Bacteria Yeast Ribosome Surface Display system Phage display Cell display Bacterium display Yeast display Mammalian cell display In vitro display Ribosome display mRNA display DNA display Phage Display A method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes Originally invented by George P. Smith in 1985 and he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest on to gene3 of filamentous phage Phage Display Further developed and improved by groups at the MRC Cambridge with Winter and McCafferty and The Scripps Research Institute with Lerner and Barbas for display of proteins like antibodies for therapeutic protein engineering. Phage display provides a direct physical link between genotype and phenotype because of the ease and rapidity of DNA sequence analysis, selected molecules can be identified quickly. The most common bacteriophages used in phage display are M13 and fd filamentous phage, though T4, T7, and λ phage have also been used. 什么是噬菌体展示 是一项筛选技术，将外源多肽或蛋白与噬菌体的衣壳蛋白融合表达，融合蛋白展示在病毒颗粒的表面，而编码该融合子的DNA则位于病毒粒子内。 使大量多肽与其DNA编码序列之间建立了直接联系，使各种靶分子（抗体、酶、细胞表面受体等）的多肽配体通过淘选得以快速鉴定。 Principles of phage display If the foreign DNA is fused to a gene coding for a viral coat protein (capsid), the foreign peptide or antibody domain can be expressed as a fusion protein with the coat protein and displayed on the surface of the phage Using a process of biopanning, one can rescue phage that display a protein that specifically binds to a target of interest. Biopanning Coating a plate with the target Incubating the library on the plate to allow phage displaying a complementary protein to the target to bind washing away non-binding phage Eluted phage that bind target Infection of bacteria with the binding phage results in phage amplificatio