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

• Published in: Analytical biochemistry Vol. 407; no. 2; pp. 237 - 240
• Main Authors: Thomas, William D., Golomb, Miriam, Smith, George P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.12.2010
• Subjects: Affinity selection; Anti-Bacterial Agents - pharmacology; Bacteriophages - metabolism; Filamentous phage libraries; Peptide Library; Peptides - chemistry; Phage display; Phage propagation; Recombination; Recombination, Genetic; Target-unrelated peptides; Tetracycline - pharmacology; Phage display; Recombination; Filamentous phage libraries; Affinity selection; Target-unrelated peptides; Phage propagation
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2010.07.037

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.