Selection biopanning

Figure 16.4 Graph depicting the percentage of lysine residues among peptides that bind to the indicated monoclonal antibodies. The peptides were isolated after affinity selection (biopanning) from a phage-displayed combinatorial peptide library. The peptides are grouped as to whether they are susceptible to formalin fixation, resulting in a loss of immunoreactivity.

Giordano RJ, Card6-Viia M, Lahden-ranta J et al. Biopanning and rapid analysis of selective interactive ligands. Nature Med 2001 7 1249-1253. 

FIGURE 16.2 Biopanning. In a typical experiment the target molecule is bound to a solid support and aspecific sites are saturated. The display library is then incubated with the target and the unbound clones are eluted, amplified, and used for two or more rounds of selection. 

The great power of biological libraries lies in the possibility to make use of biopanning [6, 7], which represents an in vitro selection process that has been employed to isolate target-binding sequences from a large pool of different molecules [8, 9]. The procedure, pioneered by Smith and others [10, 11], is performed by iterative cycles which include the binding of the library to an... 

On the basis of these considerations, this technique has become a widely used selection method to identify new binding molecules [12-17], mimotopes [18-23], or to characterize protein ligand and/or protein protein interactions [24-37]. Moreover, attempts to optimize the selection process have resulted in the setting up of new biopanning procedures, which have been performed not only on purified targets [24-37], cells [38, 39], and tissues [40] (in vitro selection), but also on living animals [41] (in vivo selection). 

This biopanning procedure, which represents the most frequently used in vitro selection method [48-59], is represented schematically in Figure 19.1. 

FIGURE 19.4 Biopanning selection through biotin-streptavidin interaction. The library is incubated with the biotinylated target in solution phages displaying the active binding peptide are captured on a streptavidin-coated Petri dish, eluted, and amplified. 

This biopanning technique has been employed to select peptide ligands that bind proteins [29, 68, 69], antibodies [30], and nucleic acid molecules [67] with moderate affinity. However, the fact that the column also retains sequences with low affinity compromises the utility of using this affinity selection to discriminate between tight or moderately binding sequences. 

FIGURE 19.6 Biopanning on a column. The target is covalently immobilized on a chromatographic support. The phage library is then applied to the column and, after an extensive washing step, bound phages are recovered by elution, amplified, and submitted to a successive selection cycle. 

FIGURE 19.8 Biopanning procedure on cells. The binding of the phage library, followed by elution and amplification steps, allows the selection of ligands directed against specific cell markers. 

Fig. 5.1. The principle of phage display is based on in vitro selection of peptides or proteins expressed at the surface of filamentous phages. In a biopanning experiment, specific binders can be captured from a library and their genes can be amplified by infection.

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