Team:Potsdam Bioware/Methods

Methods

Principle of phage display
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. It is defined as a system in which the protein and its encoding gene are physically linked. The coupling of peptides to phages and the subsequent selection of binders was first described by George P. Smith in 1985. The bacterial viruses principally used are members of the single-stranded filamentous phage family M13. After attachment to the bacterial F-pilus and entering the cells this phages stripe of their protein coat and convert their DNA in the double stranded form. Than replication, expression and assembly of phage particles occurs. About 200 phages can be produced per cell and generation. The M13 phage is a non-lytic phage, that means that they can be released from bacterial cells without causing cell death.
In M13 phage display genes from a DNA-library are usually fused to the gene III coat protein which occurs in five. The gene III protein is responsible for phage infection and releasing phage particles after production (Holliger and Riechmann, 1997; Stengele et al., 1990). Along with insertion of the gene of interest into the phage genome (McCafferty, 1990) phagemid vectors are established (Mead, 1988). Phagemids contain phage and bacterial origins of replication, the gene encoding the coat protein for fusion and a marker gene. To produce functional phages, co-infection with helper phages is required. These plasmids contain all genes used for producing phage particles. Its genome carries a mutation which leads to more efficient packaging of the phage genome of interest in relation to the helper plasmid genome.
One advantage of phage display is the rapid identification and concentration of target binding proteins through a procedure called biopanning. Bound phages are enriched by consecutive cycles of incubation, washing, amplification and selection. Therefor the target molecules are immobilized on ELISA plates or immune tubes. After an appropriate number of panning rounds, the inserts from the enriched phage can be sequenced to identify the interacting proteins.