Phagedisplay噬菌体展示技术.pptVIP

Phage Display Tu Renjun Institute of Life Sciences, Southeast University turenjun@189.cn 2011.10.13 Introduction Invented by George P. Smith in 1985. A method for the study of protein–protein, protein–peptide, and protein-DNA interactions The connection between genotype and phenotype enables large libraries of proteins to be screened and amplified in a process called in vitro selection. The most common bacteriophages used in phage display are M13 and fd filamentous phage, though T4, T7, and λ phage have also been used. General Protocol Immobilise: Target proteins or DNA sequences are immobilised to the wells of a microtiter plate. Bind: Many genetic sequences are expressed in a bacteriophage library, the added to the dish and after allowing the phage time to bind, the dish is washed. Wash: Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away. Elute: Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. Sequence: The DNA within the interacting phage contains the sequences of interacting proteins. Display System Classifications Phage: M13, f1, fdT7, T4, lamda Vector: True Phage, Phagemid Library: Random peptide library, cDNA library, antibody library, protein library M13 phage M13 phage The minor coat protein P3 attaches to the receptor at the tip of the?F pilus?of the host?E.coli. M13?plasmids?are used for many recombinant?DNA?processes, and the virus has also been studied for its uses in nanostructures and nanotechnology. Life Cycle of M13 M13 pIII pVIII Coat-Protein System pIII minor Coat-Protein Consist of 406 aa, 5 copies per phage. Located at the tail of the phage. Can display 300aa protein. pVIII major Coat-Protein 2700 copies per phage, 10% can used for display. ~200 copies of fusion proteins per phage. Proteins are usually small. High affinity abilities because of high copy numbers. Applications Determination o