A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.
Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.
Brammer
Leighanne A
LA
Department of Chemistry, Stonehill College, Easton, MA 02375, USA.
Bolduc
Benjamin
B
Kass
Jessica L
JL
Felice
Kristin M
KM
Noren
Christopher J
CJ
Hall
Marilena Fitzsimons
MF
D017104:Bacteriophage M13 / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012270:Ribosomes / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D010455:Peptides; D014764:Viral Proteins; C488880:gene II protein, Enterobacteria phage f1