Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures

Abstract

Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.

Fig. 1

Schematic diagram of alternative structures at the minus- and plus-strand origins. Functional minus- and plus-strand origins are labeled ori− and ori+, respectively; plus-strand synthesis proceeds from left to right as shown, minus-strand synthesis in the opposite direction. The symbols are not drawn to scale: the minus- and plus-strand origins span only a few dozen nucleotides each, while the Tn10-derived tetracycline resistance cassette (Tet) that disrupts the minus-strand origin in the second line spans 2774 nucleotides. The names to be used in this article for the four minus-strand origin structures are listed on the right.

Fig. 2

Recombination events giving rise to the chimeric f3-15mer clone, as inferred from its hybrid junction sequences. The chimeric clone is hypothesized to have arisen from two homologous recombination events involving double-stranded RF DNAs from the f3-15mer library itself and from a contaminant with wild-type phage fd replication origins. A. Nucleotide sequence alignments of parental genomes with the hybrid junctions of the chimeric clone. Parental nucleotides identical to those of the chimeric clone are in bold type. Junction 1 is formed by recombination between the imperfect inverted repeats (shading) that define the stem of hairpin [B], an essential element of the minus-strand origin [12]. Sequence differences bracketing the junction limit the exchange to within the 4 nucleotides marked with the bar. Because the inverted repeats flank the Tet cassette in the disrupted f3-15mer origin, the exchange inverts the Tet cassette. At Junction 2, an f3-15mer minus strand is joined near one end of its Tet cassette to a wild-type fd plus strand just upstream of its intact minus-strand origin. The hybrid junction occurs within a short string of chance sequence identities (shading); bracketing differences limit the exchange to within the 5 nucleotides marked with the bar. B. A recombinant molecule with the exchanges depicted in A would resolve during replication into the chimeric f3-15 clone. Replication origins and the Tet cassette are represented as in Fig. 1; the cross-hatched symbol represents the coding sequence for the displayed peptide. Initially, a recombinant molecule would have two same-sense oriented plus-strand origins. The next round of plus-strand synthesis starting at the left-hand origin would terminate at the right-hand origin, circularizing the unit genome in between, which corresponds to the observed chimeric clone. Subsequently, a single exchange between a chimeric clone and a non-chimeric f3-15mer clone anywhere in the 4013 nucleotides of sequence identity between the peptide coding sequence and Junction 1 would resolve at the next cycle of plus-strand synthesis into a new circular chimeric genome bearing the peptide sequence from the non-chimeric clone.

Fig. 3

Infectivity parameters of phage clones. A. Percent infectivity measured as tetracycline-resistant colony-forming units (cfu) per virion; open triangles, filled circles and open diamonds mark data-points from three independent titerings. B. Scattergram of plaque areas. C–H. Photographs of IV1–IV4, fd-tet and fd plaques, respectively.