Biased selection of propagation-related TUPs from phage display peptide libraries

Abstract

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

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“…Consequently, these peptides show a higher than expected frequency within the library [53]. By performing fewer rounds of selection, the bias can be reduced by decreasing the loss of relevant sequences [54].…”

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“…The HIAYPRH peptide (Table 4) is an illustrative example of a TUP that somehow remains overlooked by numerous research groups up until now. It was first reported by Lee [28] in 2001 as a TfR ligand but in 2007 Brammer and co-workers [29] demonstrated that it was actually a TUP and later on it was also listed as a TUP in some reviews on this topic [24][25][26]. Furthermore, 30 entries can be found in the BDB for 21 different targets, clearly supporting that it is indeed a TUP.…”

“…Secondly, phage display can be biased, and it is common to come across target unrelated peptides (TUPs) [23][24][25][26]. These motifs do not bind the actual target, but other elements in the system, mostly the polystyrene of which common labware is made, bovine serum albumin, streptavidin, antibodies and bivalent metal ions.…”

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“…However, it suffers a lot from all kinds of false positive peptides, which do not bind to or only bind to the target non-specifically. These peptides are so-called targetunrelated peptides (TUP) [37][38][39][40]. With recent advances on bioinformatics studies in this field, there are currently a series of computational resources that can help to deal with this problem [41].…”