TY - JOUR

T1 - A White Plaque, Associated with Genomic Deletion, Derived from M13KE-Based Peptide Library Is Enriched in a Target-Unrelated Manner during Phage Display Biopanning Due to Propagation Advantage

AU - Sell, Danna Kamstrup

AU - Sloth, Ane Beth

AU - Bakhshinejad, Babak

AU - Kjaer, Andreas

N1 - Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022

Y1 - 2022

N2 - The nonspecific enrichment of target-unrelated peptides during biopanning remains a major drawback for phage display technology. The commercial Ph.D.™-7 phage display library is used extensively for peptide discovery. This library is based on the M13KE vector, which carries the lacZα sequence, leading to the formation of blue plaques on IPTG-X-gal agar plates. In the current study, we report the isolation of a fast-propagating white clone (displaying WSLGYTG peptide) identified through screening against a recombinant protein. Sanger sequencing demonstrated that white plaques are not contamination from environmental M13-like phages, but derive from the library itself. Whole genome sequencing revealed that the white color of the plaques results from a large 827-nucleotide genomic deletion. The phenotypic characterization of propagation capacity through plaque count-and NGS-based competitive propagation assay supported the higher propagation rate of Ph-WSLGYTG clone compared with the library. According to our data, white plaques are likely to arise endogenously in Ph.D. libraries due to mutations in the M13KE genome and should not always be viewed as exogenous contamination. Our findings also led to the conclusion that the deletion observed here might be an ancestral mutation already present in the naïve library, which causes target-unrelated nonspecific enrichment of white clone during biopanning due to propagation advantage.

AB - The nonspecific enrichment of target-unrelated peptides during biopanning remains a major drawback for phage display technology. The commercial Ph.D.™-7 phage display library is used extensively for peptide discovery. This library is based on the M13KE vector, which carries the lacZα sequence, leading to the formation of blue plaques on IPTG-X-gal agar plates. In the current study, we report the isolation of a fast-propagating white clone (displaying WSLGYTG peptide) identified through screening against a recombinant protein. Sanger sequencing demonstrated that white plaques are not contamination from environmental M13-like phages, but derive from the library itself. Whole genome sequencing revealed that the white color of the plaques results from a large 827-nucleotide genomic deletion. The phenotypic characterization of propagation capacity through plaque count-and NGS-based competitive propagation assay supported the higher propagation rate of Ph-WSLGYTG clone compared with the library. According to our data, white plaques are likely to arise endogenously in Ph.D. libraries due to mutations in the M13KE genome and should not always be viewed as exogenous contamination. Our findings also led to the conclusion that the deletion observed here might be an ancestral mutation already present in the naïve library, which causes target-unrelated nonspecific enrichment of white clone during biopanning due to propagation advantage.

KW - Biopanning

KW - Competitive propagation

KW - Genomic deletion

KW - LacZα sequence

KW - M13KE

KW - Ph.D.™-7 peptide library

KW - Phage display

KW - Propagation-related TUP

KW - White plaque

UR - http://www.scopus.com/inward/record.url?scp=85126862972&partnerID=8YFLogxK

U2 - 10.3390/ijms23063308

DO - 10.3390/ijms23063308

M3 - Journal article

C2 - 35328728

AN - SCOPUS:85126862972

VL - 23

SP - 1

EP - 16

JO - International Journal of Molecular Sciences (Online)

JF - International Journal of Molecular Sciences (Online)

SN - 1661-6596

IS - 6

M1 - 3308

ER -