The Phage Display

Phage display technology enables in vitro selection from libraries of proteins or peptides displayed on the surface of filamentous bacteriophages. It was introduced by George P. Smith in 1985 [2] and, since then, several thousands research papers 

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Figure 4.6 The phage display scheme for the production of never-born proteins. Proline-arginine-glycine (PRG), is a substrate for thrombin, and TAG is the antibody target. (Modified from Chiarabelli et al, in press.)...

Figure 8. Selection of the phage from the phage-displayed combinatorial peptide library [9]...

The procedure for epitope mapping by the phage display method involves biopanning, amplification, assay for positive colonies, DNA isolation, and sequencing. Some steps of the procedure must be performed at the same time. Therefore, we describe the protocols in Subheadings 3.1. and 3.2. on a day-by-day manner. All steps of biopanning and phage amplification should be carried out under sterile conditions, when possible. 

Osman, A.A., Uhlig, H., Thamrn, B., Schneider-Mergener, J., Mothes, T. 1998. Use of the phage display technique for detection of epitopes recognized by polyclonal rabbit gliadin antibodies. FEBS Lett 433 103-107. 

Since its introduction, this technique has gained more and more importance in the combinatorial field. The main advantage of biological libraries, in comparison to chemical libraries, depends on the possibility of increasing remarkably the number of different molecules that can be screened at the same time. Furthermore, once the phage displaying the active sequence has been selected, it is able to replicate itself by infecting host bacterial cells successive selection and amplification cycles result in an exponential enrichment of the active peptide sequence. 

Obviously, phage display technology requires the protein to be properly folded and stable as a fusion with the coat protein. Successful display may depend on the bacterial host and on growth conditions. General strategies to improve recombinant protein expression in E.coli [36] should be transposable to the phage display context. 

A product-capture strategy was also used to select improved subtiligases. Subtiligase is a double mutant of subtilisine that catalyses the ligation of peptides. A library of >109 variants involving 25 residues of the active site was constructed on the phage-displayed enzyme. Variants that ligated a biotinylated peptide on their own ex-... 

Fig. 5.14. Strategy for the selection of glutathione-S-transferase (GST) [66], The two substrates are derivatized respectively with the calmodulin binding peptide (CBD) and a biotin (Bt). The product comprising both modules binds to the phage-displayed calmodulin (CaM) and the phage is then captured with immobilised streptavidin (Sv).

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